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To acclimate to hypoxic waterlogged conditions, the roots of wetland plants form a radial oxygen loss (ROL) barrier that can promote oxygen diffusion to the root tips. We hypothesized that the low-nitrate concentrations that occur after molecular oxygen is consumed in waterlogged soils are an environmental trigger for ROL barrier formation in rice (Oryza sativa). We previously identified 128 tissue-specific up/downregulated genes during rice ROL barrier formation. The RiceXPro database showed that many of these genes were differentially regulated in response to nitrogen deficiency. Therefore, we assessed changes in the concentrations of ionic species of nitrogen under stagnant conditions, i.e. in a nutrient solution that mimics waterlogged soil conditions, and examined the effects of an increase or decrease of nitrate in the nutrient solution on ROL barrier formation and exodermal suberization. Preventing nitrate deficiency in the stagnant nutrient solution suppressed the formation of an ROL barrier. Conversely, a decrease in nitrate strongly induced ROL barrier formation, even under aerated conditions. In parallel with ROL barrier formation, suberin lamellae formed at the exodermis. Nitrate deficiency also promoted aerenchyma formation and the enlargement of root diameters. These findings suggest that the severe decline of nitrates under waterlogged conditions is an environmental cue for exodermal suberization to form an ROL barrier in rice roots., Competing Interests: Conflict of interest. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

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 m / z ) including most plant hormones. Another technique, nanoparticle-assisted laser desorption/ionization (Nano-PALDI), can avoid matrix self-ionization using nanoparticles to assist the ionization of analytes. Here, we compared the ionization efficiencies of common plant hormones by MALDI-MS and Nano-PALDI-MS. For the comparison, we prepared a standard mix of seven plant hormones [ABA, auxin (IAA), brassinosteroid (Br), two CKs ( trans -zeatin, t Z, and 6-(γ,γ-dimethylallylamino) purine, iP), jasmonic acid, and salicylic acid (SA)], an ethylene precursor (1-aminocyclopropane-1-carboxylic acid, ACC), and a heavy hydrogen-labeled ABA (D 6 -ABA). Basic MALDI-MS detected all compounds except IAA, Br, and D 6 -ABA, while Nano-PALDI-MS detected all nine compounds. By Nano-PALDI-MS imaging (MSI), each of the abovementioned hormones and ACC were also detected in root cross sections of rice which were incubated in the hormone mix for 2 h. In the elongation zone of untreated roots, Nano-PALDI-MSI revealed high levels of ABA and CKs in the outer part of roots and much lower levels in the stele, but Br, SA, and ACC were broadly distributed in the cross section. IAA seemed to be distributed in the epidermis, cortex, and stele. Multiple-hormone imaging using Nano-PALDI-MS has great potential for investigating the roles of hormone signaling in crop development and stress responses.

Plant hormones act as important signaling molecules that regulate responses to abiotic stress as well as plant growth and development. Because their concentrations of hormones control the physiological responses in the target tissue, it is important to know the distributions and concentrations in the tissues. However, it is difficult to determine the hormone concentration on the plant tissue as a result of the limitations of conventional methods. Here, we report the first multi-imaging of two plant hormones, one of cytokinin [i.e., trans-zeatin (tZ)] and abscisic acid (ABA) using a new technology, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) imaging. Protonated signals of tZ (m/z 220.1) and ABA (m/z 265.3) were chosen on longitudinal sections of rice roots for MS imaging. tZ was broadly distributed about 40 mm behind the root apex but was barely detectable at the apex, whereas ABA was mainly detected at the root apex. Multi-imaging using MALDI-TOF-MS enabled the visualization of the localization and quantification of plant hormones. Thus, this tool is applicable to a wide range of plant species growing under various environmental conditions.

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

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 C(28) and C(30) 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., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

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., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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.

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.

The male gametophyte and tapetum play different roles during anther development although they are differentiated from the same cell lineage, the L2 layer. Until now, it has not been possible to delineate their transcriptomes due to technical difficulties in separating the two cell types. In the present study, we characterized the separated transcriptomes of the rice microspore/pollen and tapetum using laser microdissection (LM)-mediated microarray. Spatiotemporal expression patterns of 28,141 anther-expressed genes were classified into 20 clusters, which contained 3,468 (12.3%) anther-enriched genes. In some clusters, synchronous gene expression in the microspore and tapetum at the same developmental stage was observed as a novel characteristic of the anther transcriptome. Noteworthy expression patterns are discussed in connection with gene ontology (GO) categories and gene annotations, which are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation.

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.