Age-dependent analysis dissects the stepwise control of auxin-mediated lateral root development in rice.

As root elongation rates are different among each individual root, the distance from the root apices does not always reflect the age of root cells. Thus, methods for correcting variations in elongation rates are needed to accurately evaluate the root developmental process. Here, we show that modeling-based age-dependent analysis is effective for dissecting stepwise lateral root (LR) development in rice (Oryza sativa). First, we measured the increases in LR and LR primordium (LRP) numbers, diameters, and lengths in wild type and an auxin-signaling-defective mutant, which has a faster main (crown) root elongation rate caused by the mutation in the gene encoding AUXIN/INDOLE-3-ACETIC ACID protein 13 (IAA13). The longitudinal patterns of these parameters were fitted by the appropriate models and the age-dependent patterns were identified using the root elongation rates. As a result, we found that LR and LRP numbers and lengths were reduced in iaa13. We also found that the duration of the increases in LR and LRP diameters were prolonged in iaa13. Subsequent age-dependent comparisons with gene expression patterns suggest that AUXIN RESPONSE FACTOR11 (ARF11), the homolog of MONOPTEROS (MP)/ARF5 in Arabidopsis (Arabidopsis thaliana), is involved in the initiation and growth of LR(P). Indeed, the arf11 mutant showed a reduction of LR and LRP numbers and lengths. Our results also suggest that PINOID-dependent rootward-to-shootward shift of auxin flux contributes to the increase in LR and LRP diameters. Together, we propose that modeling-based age-dependent analysis is useful for root developmental studies by enabling accurate evaluation of root traits' expression.

CDPK5 and CDPK13 play key roles in acclimation to low oxygen through the control of RBOH-mediated ROS production in rice.

CALCIUM-DEPENDENT PROTEIN KINASE (CDPK) stimulates reactive oxygen species (ROS)-dependent signaling by activating RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). The lysigenous aerenchyma is a gas space created by cortical cell death that facilitates oxygen diffusion from the shoot to the root tips. Previously, we showed that RBOHH is indispensable for the induction of aerenchyma formation in rice (Oryza sativa) roots under low-oxygen conditions. Here, we showed that CDPK5 and CDPK13 localize to the plasma membrane where RBOHH functions. Mutation analysis of the serine at residues 92 and 107 of RBOHH revealed that these residues are required for CDPK5- and CDPK13-mediated activation of ROS production. The requirement of Ca2+ for CDPK5 and CDPK13 function was confirmed using in vitro kinase assays. CRISPR/Cas9-based mutagenesis of CDPK5 and/or CDPK13 revealed that the double knockout almost completely suppressed inducible aerenchyma formation, whereas the effects were limited in the single knockout of either CDPK5 or CDPK13. Interestingly, the double knockout almost suppressed the induction of adventitious root formation, which is widely conserved in vascular plants, under low-oxygen conditions. Our results suggest that CDPKs are essential for the acclimation of rice to low-oxygen conditions, and also for many other plant species conserving CDPK-targeted phosphorylation sites in RBOH homologues.

Genetic basis controlling rice plant architecture and its modification for breeding.

The shoot and root system architectures are fundamental for crop productivity. During the history of artificial selection of domestication and post-domestication breeding, the architecture of rice has significantly changed from its wild ancestor to fulfil requirements in agriculture. We review the recent studies on developmental biology in rice by focusing on components determining rice plant architecture; shoot meristems, leaves, tillers, stems, inflorescences and roots. We also highlight natural variations that affected these structures and were utilized in cultivars. Importantly, many core regulators identified from developmental mutants have been utilized in breeding as weak alleles moderately affecting these architectures. Given a surge of functional genomics and genome editing, the genetic mechanisms underlying the rice plant architecture discussed here will provide a theoretical basis to push breeding further forward not only in rice but also in other crops and their wild relatives.

Fine control of aerenchyma and lateral root development through AUX/IAA- and ARF-dependent auxin signaling.

Lateral roots (LRs) are derived from a parental root and contribute to water and nutrient uptake from the soil. Auxin/indole-3-acetic acid protein (AUX/IAA; IAA) and auxin response factor (ARF)-mediated signaling are essential for LR formation. Lysigenous aerenchyma, a gas space created by cortical cell death, aids internal oxygen transport within plants. Rice (Oryza sativa) forms lysigenous aerenchyma constitutively under aerobic conditions and increases its formation under oxygen-deficient conditions; however, the molecular mechanisms regulating constitutive aerenchyma (CA) formation remain unclear. LR number is reduced by the dominant-negative effect of a mutated AUX/IAA protein in the iaa13 mutant. We found that CA formation is also reduced in iaa13 We have identified ARF19 as an interactor of IAA13 and identified a lateral organ boundary domain (LBD)-containing protein (LBD1-8) as a target of ARF19. IAA13, ARF19, and LBD1-8 were highly expressed in the cortex and LR primordia, suggesting that these genes function in the initiation of CA and LR formation. Restoration of LBD1-8 expression recovered aerenchyma formation and partly recovered LR formation in the iaa13 background, in which LBD1-8 expression was reduced. An auxin transport inhibitor suppressed CA and LR formation, and a natural auxin stimulated CA formation in the presence of the auxin transport inhibitor. Our findings suggest that CA and LR formation are both regulated through AUX/IAA- and ARF-dependent auxin signaling. The initiation of CA formation lagged that of LR formation, which indicates that the formation of CA and LR are regulated differently by auxin signaling during root development in rice.

Targeted gene disruption of ATP synthases 6-1 and 6-2 in the mitochondrial genome of Arabidopsis thaliana by mitoTALENs.

We recently achieved targeted disruptions of cytoplasmic male sterility (CMS)-associated genes in the mitochondrial genomes of rice and rapeseed by using mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs). It was the first report of stable and heritable targeted gene modification of plant mitochondrial genomes. Here, we attempted to use mitoTALENs to disrupt two mitochondrial genes in the model plant Arabidopsis thaliana(Arabidopsis) using three different promoters and two types of TALENs. The targets were the two isoforms of the ATP synthase subunit 6 gene, atp6-1 and atp6-2. Each of these genes was successfully deleted and the mitochondrial genomes were recovered in a homoplasmic state. The nuclear genome also has a copy of atp6-1, and we were able to confirm that it was the mitochondrial gene and not the nuclear pseudogene that was knocked out. Among the three mitoTALEN promoters tried, the RPS5A promoter was the most effective. Conventional mitoTALENs were more effective than single-molecule mito-compactTALENs. Targeted mitochondrial gene deletion was achieved by crossing as well as by floral-dip transformation to introduce the mitoTALEN constructs into the nucleus. The gene disruptions were caused by large (kb-size) deletions. The ends of the remaining sequences were connected to distant loci, mostly by illegitimate homologous recombinations between repeats.

Key root traits of Poaceae for adaptation to soil water gradients.

Drought and flooding are contrasting abiotic stressors for plants. Evidence is accumulating for root anatomical traits being essential for the adaptation to drought or flooding. However, an integrated approach to comprehensively understand root anatomical traits has not yet been established. Here we analysed the root anatomical traits of 18 wild Poaceae species differing in adaptation to a range of soil water content. Regression model analyses revealed the optimal anatomical traits that were required by the plants to adapt to low or high soil water content. While the area and number of each root tissue (e.g. stele, cortex, xylem or aerenchyma) were not strongly correlated to the soil water content, the ratio of the root tissue areas (cortex to stele ratio (CSR), xylem to stele ratio (XSR) and aerenchyma to cortex ratio (ACR)) could fully explain the adaptations of the wild Poaceae species to the soil water gradients. Our results demonstrate that the optimal anatomical traits for the adaptations to soil water content can be determined by three indices (i.e. CSR, XSR and ACR), and thus we propose that these root anatomical indices can be used to improve the tolerance of crops to drought and flooding stresses.

Climate-smart crops: key root anatomical traits that confer flooding tolerance.

Plants require water, but a deficit or excess of water can negatively impact their growth and functioning. Soil flooding, in which root-zone is filled with excess water, restricts oxygen diffusion into the soil. Global climate change is increasing the risk of crop yield loss caused by flooding, and the development of flooding tolerant crops is urgently needed. Root anatomical traits are essential for plants to adapt to drought and flooding, as they determine the balance between the rates of water and oxygen transport. The stele contains xylem and the cortex contains aerenchyma (gas spaces), which respectively contribute to water uptake from the soil and oxygen supply to the roots; this implies that there is a trade-off between the ratio of cortex and stele sizes with respect to adaptation to drought or flooding. In this review, we analyze recent advances in the understanding of root anatomical traits that confer drought and/or flooding tolerance to plants and illustrate the trade-off between cortex and stele sizes. Moreover, we introduce the progress that has been made in modelling and fully automated analyses of root anatomical traits and discuss how key root anatomical traits can be used to improve crop tolerance to soil flooding.

An NADPH Oxidase RBOH Functions in Rice Roots during Lysigenous Aerenchyma Formation under Oxygen-Deficient Conditions.

Reactive oxygen species (ROS) produced by the NADPH oxidase, respiratory burst oxidase homolog (RBOH), trigger signal transduction in diverse biological processes in plants. However, the functions of RBOH homologs in rice (Oryza sativa) and other gramineous plants are poorly understood. Ethylene induces the formation of lysigenous aerenchyma, which consists of internal gas spaces created by programmed cell death of cortical cells, in roots of gramineous plants under oxygen-deficient conditions. Here, we report that, in rice, one RBOH isoform (RBOHH) has a role in ethylene-induced aerenchyma formation in roots. Induction of RBOHH expression under oxygen-deficient conditions was greater in cortical cells than in cells of other root tissues. In addition, genes encoding group I calcium-dependent protein kinases (CDPK5 and CDPK13) were strongly expressed in root cortical cells. Coexpression of RBOHH with CDPK5 or CDPK13 induced ROS production in Nicotiana benthamiana leaves. Inhibitors of RBOH activity or cytosolic calcium influx suppressed ethylene-induced aerenchyma formation. Moreover, knockout of RBOHH by CRISPR/Cas9 reduced ROS accumulation and inducible aerenchyma formation in rice roots. These results suggest that RBOHH-mediated ROS production, which is stimulated by CDPK5 and/or CDPK13, is essential for ethylene-induced aerenchyma formation in rice roots under oxygen-deficient conditions.

OsPIN2, which encodes a member of the auxin efflux carrier proteins, is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice.

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport-related mutants, Ospin-formed2-1 (Ospin2-1) and Ospin2-2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5-driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N-1-naphthylphthalamic acid and Ospin2-1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2-1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild-type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild-type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.

Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots.

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.

Ethylene-dependent aerenchyma formation in adventitious roots is regulated differently in rice and maize.

In roots of gramineous plants, lysigenous aerenchyma is created by the death and lysis of cortical cells. Rice (Oryza sativa) constitutively forms aerenchyma under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. However, maize (Zea mays) develops aerenchyma only under oxygen-deficient conditions. Ethylene is involved in lysigenous aerenchyma formation. Here, we investigated how ethylene-dependent aerenchyma formation is differently regulated between rice and maize. For this purpose, in rice, we used the reduced culm number1 (rcn1) mutant, in which ethylene biosynthesis is suppressed. Ethylene is converted from 1-aminocyclopropane-1-carboxylic acid (ACC) by the action of ACC oxidase (ACO). We found that OsACO5 was highly expressed in the wild type, but not in rcn1, under aerobic conditions, suggesting that OsACO5 contributes to aerenchyma formation in aerated rice roots. By contrast, the ACO genes in maize roots were weakly expressed under aerobic conditions, and thus ACC treatment did not effectively induce ethylene production or aerenchyma formation, unlike in rice. Aerenchyma formation in rice roots after the initiation of oxygen-deficient conditions was faster and greater than that in maize. These results suggest that the difference in aerenchyma formation in rice and maize is due to their different mechanisms for regulating ethylene biosynthesis.

Transcript profiles in cortical cells of maize primary root during ethylene-induced lysigenous aerenchyma formation under aerobic conditions.

BACKGROUND AND AIMS: Internal aeration is important for plants to survive during periods of waterlogging, and the ability to form aerenchyma contributes by creating a continuous gas space between the shoots and the roots. Roots of maize (Zea mays) react to prolonged waterlogging by forming aerenchyma in root cortical cells by programmed cell death (PCD) in response to ethylene. The aim of this study was to understand the molecular mechanisms of ethylene-induced aerenchyma formation by identifying genes that are either up- or downregulated by ethylene treatment in maize root cortical cells. METHODS: Three-day-old maize seedlings were treated with ethylene for several hours under aerobic conditions. Cortical cells were isolated from the primary roots using laser microdissection (LM), and transcript profiles with and without ethylene treatment were compared by microarray. In addition, the effect on ethylene-induced aerenchyma formation of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, was examined in order to assess the involvement of reactive oxygen species (ROS). KEY RESULTS: A total of 223 genes were identified whose transcript levels were significantly increased or decreased by ethylene treatment in root cortical cells under aerobic conditions. Subsequent tissue-specific quantitative reverse-transcription PCR analyses revealed that ethylene increased the transcript levels of genes related to ethylene signalling in all of the root tissues examined (stelar cells, cortical cells and outer cell layers), whereas it increased the transcript levels of genes related to cell wall modification and proteolysis specifically in the cortical cells. DPI treatment inhibited the ethylene-induced aerenchyma formation and suppressed expression of some cell wall modification-related genes. CONCLUSIONS: Several genes related to cell wall modification and proteolysis are specifically up- or downregulated in cortical cells during lysigenous aerenchyma formation under aerobic conditions with ethylene treatment. The results suggest that ethylene is perceived in stelar cells, cortical cells and outer cell layers in the maize primary root, and that the cortical cell-specific PCD is controlled downstream of ethylene perception through subsequent gene expression, which is partly regulated by ROS, in the cortical cells.

The MET1b gene encoding a maintenance DNA methyltransferase is indispensable for normal development in rice.

While Arabidopsis bears only one MET1 gene encoding the DNA methyltransferase that is mainly responsible for maintaining CG methylation after DNA replication, rice carries two MET1 genes, MET1a and MET1b, expressed in actively replicating and dividing cells, and MET1b is more abundantly expressed than is MET1a. A met1a null mutant displayed no overt phenotypes, implying that MET1b must play a major role in the maintenance DNA methylation. Here, we employed two met1b null mutants, generated by homologous recombination-mediated knock-in targeting and insertion of endogenous retrotransposon Tos17. These MET1a/MET1a met1b/met1b homozygotes exhibited abnormal seed phenotypes, which is associated with either viviparous germination or early embryonic lethality. They also displayed decreased levels of DNA methylation at repetitive CentO sequences and at the FIE1 gene locus in the embryos. In addition, independently isolated knock-in-targeted plants, in which the promoterless GUS reporter gene was fused with the endogenous MET1b promoter, showed the reproducible, dosage-dependent, and spatiotemporal expression patterns of GUS. The genotyping analysis of selfed progeny of heterozygous met1a met1b null mutants indicated that weakly active MET1a seems to serve as a genetic backup mechanism in rice met1b gametophytes, although the stochastic and uncoordinated activation of epigenetic backup mechanisms occurred less efficiently in the met1b homozygotes of rice than in the met1 homozygotes of Arabidopsis. Moreover, passive depletion of CG methylation during the postmeiotic DNA replication in the haploid nuclei of the met1a met1b gametophytes in rice results in early embryonic lethality. This situation somewhat resembles that of the met1 gametophytes in Arabidopsis.

Strigolactone and cytokinin act antagonistically in regulating rice mesocotyl elongation in darkness.

Strigolactones (SLs) are a group of phytohormones that control plant growth and development including shoot branching. Previous studies of the phenotypes of SL-related rice (Oryza sativa) dwarf (d) mutants demonstrated that SLs inhibit mesocotyl elongation by controlling cell division. Here, we found that the expression of cytokinin (CK)-responsive type-A RESPONSE REGULATOR (RR) genes was higher in d10-1 and d14-1 mutants than in the wild type. However, CK levels in mesocotyls of the d mutants were not very different from those in the wild type. On the other hand, application of a synthetic CK (kinetin) enhanced mesocotyl elongation in the d mutants and the wild type. d10-1 and d14-1 mesocotyls were more sensitive to CK than wild-type mesocotyls, suggesting that the up-regulation of the CK-responsive type-A RR genes and the higher elongation of mesocotyls in the d mutants are mainly due to the increased sensitivity of the d mutants to CK. Co-treatment with kinetin and a synthetic SL (GR24) confirmed the antagonistic functions of SL and CK on mesocotyl elongation. OsTCP5, which encodes a transcription factor belonging to the cell division-regulating TCP family, was also regulated by SL and CK and its expression was negatively correlated with mesocotyl length. These findings suggest that OsTCP5 contributes to the SL- and CK-controlled mesocotyl elongation in darkness.

Biochemical and molecular characterization of rice (Oryza sativa†L.) roots forming a barrier to radial oxygen loss.

The formation of a barrier to radial oxygen (O2 ) loss (ROL) in the root is an important adaptation of plants to root flooding, but the biochemical changes in plant roots where the barrier is formed are unclear. In this study, we analysed metabolic profiles and gene expression profiles in roots of rice (Oryza sativa L.) plants grown under stagnant deoxygenated conditions, which induce suberization in the outer cell layers of the roots and formation of barrier to ROL. Under these conditions, two distinctive biochemical features of the roots were the accumulations of malic acid and very long chain fatty acids (VLCFAs). We also showed that the expressions of some genes encoding plastid-localized enzymes, which convert malic acid to acetyl coenzyme A (AcCoA), were simultaneously up-regulated under stagnant conditions. The expression levels of these genes in specific root tissues isolated by laser microdissection suggested that malic acid is converted to AcCoA predominantly in the plastids in the outer cell layers of rice roots. We propose that the physiological role of malic acid accumulation in rice roots grown under stagnant conditions is to provide a substrate for the biosynthesis of fatty acids, which, in turn, are used in the biosynthesis of suberin.

Ethylene and reactive oxygen species are involved in root aerenchyma formation and adaptation of wheat seedlings to oxygen-deficient conditions.

Exposing plants to hypoxic conditions greatly improves their anoxic stress tolerance by enhancing the activities of glycolysis and fermentation in roots. Ethylene may also be involved in these adaptive responses because its synthesis is increased in roots under hypoxic conditions. Here it is reported that pre-treatment of wheat seedlings with an ethylene precursor, 1-aminocyclopropanecarboxylic acid (ACC), enhanced accumulation of ethylene in the roots of wheat seedlings, and enhanced their tolerance of oxygen-deficient conditions through increasing the expression of genes encoding ethanol fermentation enzymes, alcohol dehydrogenase and pyruvate decarboxylase, in the roots. Lysigenous aerenchyma formation in root was induced by ACC pre-treatment and was further induced by growth under oxygen-deficient conditions. ACC pre-treatment increased the expression of three genes encoding respiratory burst oxidase homologue (a plant homologue of gp91(phox) in NADPH oxidase), which has a role in the generation of reactive oxygen species (ROS), in roots of seedlings. Co-treatment with ACC and an NADPH oxidase inhibitor, diphenyleneiodonium, partly suppressed the ACC-induced responses. These results suggest that ethylene and ROS are involved in adaptation of wheat seedlings to oxygen-deficient conditions through controlling lysigenous aerenchyma formation and the expression of genes encoding ethanol fermentation enzymes.

Microarray analysis of laser-microdissected tissues indicates the biosynthesis of suberin in the outer part of roots during formation of a barrier to radial oxygen loss in rice (Oryza sativa).

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.

Root anatomical plasticity contributes to the different adaptive responses of two Phragmites species to water-deficit and low-oxygen conditions.

The runner reed (Phragmites japonica ) is the dominant species on riverbanks, whereas the common reed (Phragmites australis ) thrives in continuously flooded areas. Here, we aimed to identify the key root anatomical traits that determine the different adaptative responses of the two Phragmites species to water-deficit and low-oxygen conditions. Growth measurements revealed that P . japonica tolerated high osmotic conditions, whereas P . australis preferred low-oxygen conditions. Root anatomical analysis revealed that the ratios of the cortex to stele area and aerenchyma (gas space) to cortex area in both species increased under low-oxygen conditions. However, a higher ratio of cortex to stele area in P . australis resulted in a higher ratio of aerenchyma to stele, which includes xylem vessels that are essential for water and nutrient uptakes. In contrast, a lower ratio of cortex to stele area in P . japonica could be advantageous for efficient water uptake under high-osmotic conditions. In addition to the ratio of root tissue areas, rigid outer apoplastic barriers composed of a suberised exodermis may contribute to the adaptation of P . japonica and P . australis to water-deficit and low-oxygen conditions, respectively. Our results suggested that root anatomical plasticity is essential for plants to adapt and respond to different soil moisture levels.