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To acclimate to waterlogged conditions, wetland plants form a barrier to radial oxygen loss (ROL) that can enhance oxygen transport to the root apex. We hypothesized that one or more hormones are involved in the induction of the barrier and searched for such hormones in rice. We previously identified 98 genes that were tissue-specifically upregulated during ROL barrier formation in rice. The RiceXPro database showed that most of these genes were highly enhanced by exogenous abscisic acid (ABA). We then examined the effect of ABA on ROL barrier formation by using an ABA biosynthesis inhibitor (fluridone, FLU), by applying exogenous ABA and by examining a mutant with a defective ABA biosynthesis gene (osaba1). FLU suppressed barrier formation in a stagnant solution that mimics waterlogged soil. Under aerobic conditions, rice does not naturally form a barrier, but 24 h of ABA treatment induced barrier formation. osaba1 did not form a barrier under stagnant conditions, but the application of ABA rescued the barrier. In parallel with ROL barrier formation, suberin lamellae formed in the exodermis. These findings strongly suggest that ABA is an inducer of suberin lamellae formation in the exodermis, resulting in an ROL barrier formation in rice.

Internal aeration is crucial for root growth under waterlogged conditions. Many wetland plants have a structural barrier that impedes oxygen leakage from the basal part of roots called a radial oxygen loss (ROL) barrier. ROL barriers reduce the loss of oxygen transported via the aerenchyma to the root tips, enabling long-distance oxygen transport for cell respiration at the root tip. Because the root tip does not have an ROL barrier, some of the transferred oxygen is released into the waterlogged soil, where it oxidizes and detoxifies toxic substances (e.g., sulfate and Fe2+) around the root tip. ROL barriers are located at the outer part of roots (OPRs). Their main component is thought to be suberin. Suberin deposits may block the entry of potentially toxic compounds in highly reduced soils. The amount of ROL from the roots depends on the strength of the ROL barrier, the length of the roots, and environmental conditions, which causes spatiotemporal changes in the root system’s oxidization pattern. We summarize recent achievements in understanding how ROL barrier formation is regulated and discuss opportunities for breeding waterlogging-tolerant crops.

Plant hormones can act in synergistic and antagonistic ways in response to biotic and abiotic stresses and in plant growth and development. Thus, a technique is needed to simultaneously determine the distributions and concentrations of several plant hormones. Previously, we reported that localizations of two plant hormones [cytokinin (CK) and abscisic acid (ABA)] can be simultaneously visualized in a plant tissue using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). In MALDI-MS, however, self-ionization of an organic matrix occasionally interferes with ionizations of small molecules (

Background Detailed datasets containing root system and its architecture in soil are required to improve understanding of resource capture by roots. However, most of the root study methods have paid little attention to make and preserve whole root specimens. This study introduces root system sampling equipment that makes the entire root specimen with minimum impairment and without displacement of the spatial arrangement of the root system in root boxes. The objectives are to assess: whether the equipment can rapidly sample the entire root system; whether root surface area is measurable from a scanned digital image of the root specimen; and whether staining of the entire root specimens would provide multidimensional visual information on the interaction between soil and physiological function of root system architecture (RSA). For validation, we examined the root response of two soybean cultivars to arbuscular mycorrhizal (AM) inoculation and the effect of waterlogging stress on the physiological activity of buckwheat RSA. Results The root boxes allowed soybean and buckwheat plants to grow uniformly across the replications. Both species showed significant differences between cultivars and/or among treatments in shoot and root traits. The equipment enabled to sample the whole-root specimens of soybean and buckwheat, where the tips of the fine roots were alive (diameter Conclusions The present method realized: fast and accurate production of the whole root specimen and precise calculation of the specimens’ root surface area. Moreover, staining of the root specimens enabled analyzing the interaction between soil and physiological function of RSA. The evaluation of root traits, using our methods, will contribute to developing agronomic management and breeding program for sustainable food production.

Laser microdissection (LM) is a rapid, easy, one-step, and efficient method of conserving and isolating single cell or cell clusters from fixed tissue sections under direct microscopic visualization. LM is currently the only method that can be used to isolate homogeneous cells from heterogeneous tissue. This method was first developed for tumor analysis. Multiple generations of LM instruments are available on the market. For instance, the Veritas™ microdissection combines the LM system based on the IR with UV laser cutting. The desired cells can be harvested specific and wanted cells can be harvested directly by cutting target cells abroad from unwanted cells. The extracted cells are then utilized in various disciplines of experimental and clinical biology, such as genomic analysis and proteomics. LM is a an efficient and powerful tool that facilitates the analysis of small amounts of molecules isolated from a complex tissue, thus offering new opportunities to understand physiological and fundamental processes. Here, we describe a method of preparing paraffin sections of maize roots for laser microdissection to three parts (stele, cortex and outer layers) by LM using a microwave embedding method. This approach allows RNA to be extracted from each type of tissue separately. The RNA integrity of our samples following LM ranged between 7 and 7.2, indicating that this RNA can be reliably used for further analysis. The work reported in this paper highlights how advances in protocols and methods have made LM a powerful and sometimes essential tool for genomic and proteomic analyses of tiny amounts of biomolecules extracted from a few cells isolated from a complex tissue in their physiological context, thus offering a new pathway to understand fundamental, physiological and/or patho-physiological processes.

Plant hormones can act in synergistic and antagonistic ways in response to biotic and abiotic stresses and in plant growth and development. Thus, a technique is needed to simultaneously determine the distributions and concentrations of several plant hormones. Previously, we reported that localizations of two plant hormones [cytokinin (CK) and abscisic acid (ABA)] can be simultaneously visualized in a plant tissue using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). In MALDI-MS, however, self-ionization of an organic matrix occasionally interferes with ionizations of small molecules (500

Passage cells are frequently found in the exodermis and the endodermis of the roots. Because passage cells lack an apoplastic diffusion barrier, they are thought to provide pathways for the transport of nutrients and the entrance of endomycorrhizal fungi. Exodermal passage cells possess Casparian strips but not suberin lamellae. So far, exodermal passage cells have not been associated with a particular internal structure. In some wetland plants, the outer part of the root (i.e., epidermis, exodermis, and sclerenchyma) of emerging lateral root primordia has an oxygen leaky zone called a window. The exodermis at the window site also lacks suberin lamellae, but it remains unclear whether the exodermis at the window site also lacks Casparian strips. Here, we report that several of the exodermal cells in the window of Echinochloa crus-galli grown under aerated or deoxygenated stagnant agar nutrient solution also lack lignin, which is a major constituent of Casparian strips. The sclerenchyma cells that form part of the window also lacked lignin deposits. Sites at which lateral root primordia developed were highly permeable to an apoplastic tracer (periodic acid). These observations indicate that windows consist of a novel type of passage cell at the exodermis that lacks lignin as well as suberin lamellae.

Passage cells are frequently found in the exodermis and the endodermis of the roots. Because passage cells lack an apoplastic diffusion barrier, they are thought to provide pathways for the transport of nutrients and the entrance of endomycorrhizal fungi. Exodermal passage cells possess Casparian strips but not suberin lamellae. So far, exodermal passage cells have not been associated with a particular internal structure. In some wetland plants, the outer part of the root (i.e., epidermis, exodermis, and sclerenchyma) of emerging lateral root primordia has an oxygen leaky zone called a window. The exodermis at the window site also lacks suberin lamellae, but it remains unclear whether the exodermis at the window site also lacks Casparian strips. Here, we report that several of the exodermal cells in the window of Echinochloa crus-galli grown under aerated or deoxygenated stagnant agar nutrient solution also lack lignin, which is a major constituent of Casparian strips. The sclerenchyma cells that form part of the window also lacked lignin deposits. Sites at which lateral root primordia developed were highly permeable to an apoplastic tracer (periodic acid). These observations indicate that windows consist of a novel type of passage cell at the exodermis that lacks lignin as well as suberin lamellae.

Root growth into hypoxic or anoxic waterlogged soil relies on internal aeration in plants. The plant hormone ethylene helps adapt to waterlogging by inducing the formation of aerenchyma, which provides a low-resistance pathway for the transport of oxygen from the shoot to the root apex. Waterlogging-susceptible crops including barley start to form aerenchyma after suffering waterlogging stress. But waterlogging can be fatal if aerenchyma formation is not fast enough. Here, we investigated whether pre-treating barley with ethephon, an ethylene-releasing agrochemical, could improve its tolerance to mimicked waterlogging conditions (using stagnant deoxygenated agar nutrient solution). In barley growing in aerated nutrient solution, ethephon treatment enhanced aerenchyma formation at the root tips and induced the development of shorter and shallower roots. Pre-treating barley leaves also delayed waterlogging-caused whiting and increased the percentages of viable root-tips under waterlogging conditions. However, the pretreatment did not noticeably increase fresh weight or shoot length. Further studies are needed to optimize ethephon treatment conditions to improve barley production under waterlogged conditions.

Enhancement of oxygen transport from shoot to root tip by the formation of aerenchyma and also a barrier to radial oxygen loss (ROL) in roots is common in waterlogging- tolerant plants.Zea nicaraguensis (teosinte),a wild relative of maize (Zea mays ssp. mays), grows in waterlogged soils. We investigated the formation of aerenchyma and ROL barrier induction in roots of Z. nicaraguensis, in compari- son with roots of maize (inbred line Mi29), in a pot soil system and in hydroponics. Furthermore, depositions of suberin in the exodermis/hypodermis and lignin in the epi- dermis of adventitious roots of Z. nicaraguensis and maize grown in aerated or stagnant deoxygenated nutrient solu- tion were studied. Growth of maize was more adversely affected by low oxygen in the root zone (waterlogged soil or stagnant deoxygenated nutrient solution) compared with Z. nicaraguensis. In stagnant deoxygenated solution, Z. nicaraguensis was superior to maize in transporting oxygen from shoot base to root tip due to formation of larger aerenchyma and a stronger barrier to ROL in adventitious roots. The relationships between the ROL barrier formation and suberin and lignin depositions in roots are discussed. The ROL barrier, in addition to aerenchyma, would contribute to the waterlogging tolerance of Z. nicaraguensis.

In flowering plants, the male gametophyte, the pollen, develops in the anther. Complex patterns of gene expression in both the gametophytic and sporophytic tissues of the anther regulate this process. The gene expression profiles of the microspore/pollen and the sporophytic tapetum are of particular interest. In this study, a microarray technique combined with laser microdissection (44K LM-microarray) was developed and used to characterize separately the transcriptomes of the microspore/pollen and tapetum in rice. Expression profiles of 11 known tapetum specific-genes were consistent with previous reports. Based on their spatial and temporal expression patterns, 140 genes which had been previously defined as anther specific were further classified as male gametophyte specific (71 genes, 51%), tapetum-specific (seven genes, 5%) or expressed in both male gametophyte and tapetum (62 genes, 44%). These results indicate that the 44K LM-microarray is a reliable tool to analyze the gene expression profiles of two important cell types in the anther, the microspore/pollen and tapetum.

Ethylene plays a central role in morphological and anatomical acclimations of plants to soil waterlogging. To acclimate to waterlogging, roots of wetland species, and even some crops, form aerenchyma. Aerenchyma is large interconnected gas spaces that connect from shoot to near the tips of roots. Oxygen moves via diffusion in the root aerenchyma. In many wetland species, a barrier to radial oxygen loss (ROL) that greatly reduces oxygen leakage from basal parts, further enhances oxygen diffusion to the apex. Two main types of aerenchyma are recognized: schizogenous and lysigenous aerenchymas. Schizogenous aerenchyma is formed by cell separation without cell death. Lysigenous aerenchyma results from programmed cell death (PCD). Ethylene accumulation in plants during waterlogging induces lysigenous aerenchyma, although many wetland species form some aerenchyma even in drained conditions. The ethylene accumulation may result in the activation of G-proteins and other signal transduction cascades, of which increased cytosolic Ca 2+ is a component. Finally, PCD leading to selective cell degradation occurs in the root cortex, with cell wall degrading enzymes, such as cellulase and pectinase, also being activated. On the other hand, ethylene does not appear to induce the barrier to ROL, at least in roots of rice. Thus, induction of the barrier to ROL and formation of aerenchyma appear to be regulated by different signals. Further elucidation of signal transduction pathways initiated by ethylene sensing, as well as non-ethylene dependant responses, and of the genes regulated to control formation of the barrier to ROL and aerenchyma, should be priority areas for future research on plant tolerance of waterlogging.

ハードパン破砕を伴った植林法のための適種選抜はハードパンが分布する乾燥地での植林の成功に重要である．ハードパン破砕による成長改善には種間差があり，Eucalyptus camaldulensisで改善されるが，Acacia aneuraでは改善されない．この原因として，ハードパン破砕により土壌水分状態が改善された深い土層を水源とする能力の種間差が予想されていた．本研究では，ハードパン破砕区と非破砕区（自然状態）における灌漑前後の深度別の土壌体積含水率の測定と，土壌と植林木からの抽出水，灌漑水および井戸水の酸素安定同位体比の分析により，2樹種の主な水源の深さを推定した．非破砕区では両樹種ともに厚いハードパン層のある深度20 cmよりも浅い土層を主な水源としており，灌漑前のこの土層は乾燥していた．一方，灌漑前にも破砕区の深度100 cmより深い土層は湿潤状態にあった．破砕区のE.camaldulensisはこの深度100 cmよりも深い土層の水を主な水源としていた．しかし，A.aneuraは破砕区でも深度100 cmより浅い土層を主な水源としており，深い土層の水を有効に利用できていなかった．本研究により深い土層（>100 cm）の水を主な水源として利用できる樹種はハードパン破砕により成長改善されることが推察できた．

Internal aeration is crucial for root growth in waterlogged soil. A barrier to radial oxygen loss (ROL) can enhance long-distance oxygen transport via the aerenchyma to the root tip; a higher oxygen concentration at the apex enables root growth into anoxic soil. The ROL barrier is formed within the outer part of roots (OPR). Suberin and/or lignin deposited in cell walls are thought to contribute to the barrier, but it is unclear which compound is the main constituent. This study describes gene expression profiles during ROL barrier formation in rice roots to determine the relative responses of suberin and/or lignin biosyntheses for the barrier. OPR tissues were isolated by laser microdissection and their transcripts were analysed by microarray. A total of 128 genes were significantly up- or downregulated in the OPR during the barrier formation. Genes associated with suberin biosynthesis were strongly upregulated, whereas genes associated with lignin biosynthesis were not. By an ab initio analysis of the promoters of the upregulated genes, the putative cis-elements that could be associated with transcription factors, WRKY, AP2/ERF, NAC, bZIP, MYB, CBT/DREB, and MADS, were elucidated. They were particularly associated with the expression of transcription factor genes containing WRKY, AP2, and MYB domains. A semiquantitative reverse-transcription PCR analysis of genes associated with suberin biosynthesis (WRKY, CYP, and GPAT) confirmed that they were highly expressed during ROL barrier formation. Overall, these results suggest that suberin is a major constituent of the ROL barrier in roots of rice.

Summary Suberin is a complex polymer composed of aliphatic and phenolic compounds. It is a constituent of apoplastic plant interfaces. In many plant species, including rice (Oryza sativa), the hypodermis in the outer part of roots forms a suberized cell wall (the Casparian strip and/or suberin lamellae), which inhibits the flow of water and ions and protects against pathogens. To date, there is no genetic evidence that suberin forms an apoplastic transport barrier in the hypodermis. We discovered that a rice reduced culm number1 (rcn1) mutant could not develop roots longer than 100 mm in waterlogged soil. The mutated gene encoded an ATP-binding cassette (ABC) transporter named RCN1/OsABCG5. RCN1/OsABCG5 gene expression in the wild type was increased in most hypodermal and some endodermal roots cells under stagnant deoxygenated conditions. A GFP-RCN1/OsABCG5 fusion protein localized at the plasma membrane of the wild type. Under stagnant deoxygenated conditions, well suberized hypodermis developed in wild types but not in rcn1 mutants. Under stagnant deoxygenated conditions, apoplastic tracers (periodic acid and berberine) were blocked at the hypodermis in the wild type but not in rcn1, indicating that the apoplastic barrier in the mutant was impaired. The amount of the major aliphatic suberin monomers originating from C28 and C30 fatty acids or ω-OH fatty acids was much lower in rcn1 than in the wild type. These findings suggest that RCN1/OsABCG5 has a role in the suberization of the hypodermis of rice roots, which contributes to formation of the apoplastic barrier.

Enhancement of oxygen transport from shoot to root tip by the formation of aerenchyma and also a barrier to radial oxygen loss (ROL) in roots is common in waterlogging-tolerant plants. Zea nicaraguensis (teosinte), a wild relative of maize (Zea mays ssp. mays), grows in waterlogged soils. We investigated the formation of aerenchyma and ROL barrier induction in roots of Z. nicaraguensis, in comparison with roots of maize (inbred line Mi29), in a pot soil system and in hydroponics. Furthermore, depositions of suberin in the exodermis/hypodermis and lignin in the epidermis of adventitious roots of Z. nicaraguensis and maize grown in aerated or stagnant deoxygenated nutrient solution were studied. Growth of maize was more adversely affected by low oxygen in the root zone (waterlogged soil or stagnant deoxygenated nutrient solution) compared with Z. nicaraguensis. In stagnant deoxygenated solution, Z. nicaraguensis was superior to maize in transporting oxygen from shoot base to root tip due to formation of larger aerenchyma and a stronger barrier to ROL in adventitious roots. The relationships between the ROL barrier formation and suberin and lignin depositions in roots are discussed. The ROL barrier, in addition to aerenchyma, would contribute to the waterlogging tolerance of Z. nicaraguensis.

• To adapt to waterlogging in soil, some gramineous plants, such as maize (Zea mays), form lysigenous aerenchyma in the root cortex. Ethylene, which is accumulated during waterlogging, promotes aerenchyma formation. However, the molecular mechanism of aerenchyma formation is not understood. • The aim of this study was to identify aerenchyma formation-associated genes expressed in maize roots as a basis for understanding the molecular mechanism of aerenchyma formation. Maize plants were grown under waterlogged conditions, with or without pretreatment with an ethylene perception inhibitor 1-methylcyclopropene (1-MCP), or under aerobic conditions. Cortical cells were isolated by laser microdissection and their mRNA levels were examined with a microarray. • The microarray analysis revealed 575 genes in the cortical cells, whose expression was either up-regulated or down-regulated under waterlogged conditions and whose induction or repression was suppressed by pretreatment with 1-MCP. • The differentially expressed genes included genes related to the generation or scavenging of reactive oxygen species, Ca(2+) signaling, and cell wall loosening and degradation. The results of this study should lead to a better understanding of the mechanism of root lysigenous aerenchyma formation.

Background and aims Many wetland species form aerenchyma and a barrier to radial O(2) loss (ROL) in roots. These features enhance internal O(2) diffusion to the root apex. Barrier formation in rice is induced by growth in stagnant solution, but knowledge of the dynamics of barrier induction and early anatomical changes was lacking. Methods ROL barrier induction in short and long roots of rice (Oryza sativa L. 'Nipponbare') was assessed using cylindrical root-sleeving O(2) electrodes and methylene blue indicator dye for O(2) leakage. Aerenchyma formation was also monitored in root cross-sections. Microstructure of hypodermal/exodermal layers was observed by transmission electron microscopy (TEM). Key results In stagnant medium, barrier to ROL formation commenced in long adventitious roots within a few hours and the barrier was well formed within 24 h. By contrast, barrier formation took longer than 48 h in short roots. The timing of enhancement of aerenchyma formation was the same in short and long roots. Comparison of ROL data and subsequent methylene blue staining determined the apparent ROL threshold for the dye method, and the dye method confirmed that barrier induction was faster for long roots than for short roots. Barrier formation might be related to deposition of new electron-dense materials in the cell walls at the peripheral side of the exodermis. Histochemical staining indicated suberin depositions were enhanced prior to increases in lignin. Conclusions As root length affected formation of the barrier to ROL, but not aerenchyma, these two acclimations are differentially regulated in roots of rice. Moreover, ROL barrier induction occurred before histochemically detectable changes in putative suberin and lignin deposits could be seen, whereas TEM showed deposition of new electron-dense materials in exodermal cell walls, so structural changes required for barrier functioning appear to be more subtle than previously described.

Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4–6 days to about 5 h by using a recently developed microwave method; (2) the time of floating sections from ~10 min to less than 5 min, (3) the drying time from ~12 to 1 h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.

Co-expression networks systematically constructed from large-scale transcriptome data reflect the interactions and functions of genes with similar expression patterns and are a powerful tool for the comprehensive understanding of biological events and mining of novel genes. In Arabidopsis (a model dicot plant), high-resolution co-expression networks have been constructed from very large microarray datasets and these are publicly available as online information resources. However, the available transcriptome data of rice (a model monocot plant) have been limited so far, making it difficult for rice researchers to achieve reliable co-expression analysis. In this study, we performed co-expression network analysis by using combined 44 K agilent microarray datasets of rice, which consisted of 33 laser microdissection (LM)-microarray datasets of anthers, and 143 spatiotemporal transcriptome datasets deposited in RicexPro. The entire data of the rice co-expression network, which was generated from the 176 microarray datasets by the Pearson correlation coefficient (PCC) method with the mutual rank (MR)-based cut-off, contained 24,258 genes and 60,441 genes pairs. Using these datasets, we constructed high-resolution co-expression subnetworks of two specific biological events in the anther, "meiosis" and "pollen wall synthesis". The meiosis network contained many known or putative meiotic genes, including genes related to meiosis initiation and recombination. In the pollen wall synthesis network, several candidate genes involved in the sporopollenin biosynthesis pathway were efficiently identified. Hence, these two subnetworks are important demonstrations of the efficiency of co-expression network analysis in rice. Our co-expression analysis included the separated transcriptomes of pollen and tapetum cells in the anther, which are able to provide precise information on transcriptional regulation during male gametophyte development in rice. The co-expression network data presented here is a useful resource for rice researchers to elucidate important and complex biological events.