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.

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.

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.

Asymmetric auxin distribution establishes a contrasting pattern of aerenchyma formation in the nodal roots of Zea nicaraguensis during gravistimulation.

Auxin distribution is essential for determining root developmental patterns. The formation of lateral roots and constitutive aerenchyma, which is a gas space developed through cell death, is regulated by auxin in rice (Oryza sativa). However, it is unclear whether the involvement of auxin in constitutive aerenchyma formation is conserved in other species. In this study, we found that constitutive aerenchyma formation was regulated by auxin in the nodal roots of Zea nicaraguensis, a wild relative of maize (Zea mays ssp. mays) grown naturally on frequently flooded coastal plains. Subsequent gravistimulation (root rotation) experiments showed opposite patterns of aerenchyma and lateral root formation. Lateral root formation on the convex side of rotated roots is known to be stimulated by a transient increase in auxin level in the pericycle. We found that aerenchyma formation was accelerated in the cortex on the concave side of the rotated nodal roots of Z. nicaraguensis. A cortex-specific expression analysis of auxin-responsive genes suggested that the auxin level was higher on the concave side than on the convex side. These results suggest that asymmetric auxin distribution underlies the regulation of aerenchyma and lateral root formation in the nodal roots of Z. nicaraguensis. As aerenchyma reduces the respiratory cost of the roots, constitutive aerenchyma on the concave side of the nodal root may balance resource allocation, thereby contributing to the uptake of water and nutrients by newly formed lateral roots. Our study provides insights into auxin-dependent asymmetric root patterning such as that of gravistimulation and hydropatterning response.

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.

Modeling-based age-dependent analysis reveals the net patterns of ethylene-dependent and -independent aerenchyma formation in rice and maize roots.

Plants require oxygen for the functioning of roots, and thus the establishment of a long-distance diffusion path from above-water tissues to the submerged roots is essential to survive flooding. Rice (Oryza sativa) constitutively forms aerenchyma (gas spaces) under aerobic conditions, and induces its formation in response to low-oxygen conditions. Constitutive aerenchyma formation in rice roots is regulated by the phytohormone auxin, whereas ethylene stimulates inducible aerenchyma formation. However, the net patterns of the ethylene-dependent and -independent (auxin-dependent) aerenchyma formation remain unclear. In the present study, we used a modeling approach to determine age-dependent aerenchyma formation in the wild-type rice and reduced culm number 1 mutant, in which ethylene production is reduced, to reveal the net patterns of ethylene-dependent and -independent aerenchyma formation. Subsequent comparison of age-dependent aerenchyma formation between rice and maize roots suggested that more rapid induction of ethylene-dependent aerenchyma formation and more aerenchyma in rice roots are essential to achieve efficient oxygen diffusion under low-oxygen conditions. Moreover, rice roots showed rapid increase in the expression levels of ethylene biosynthesis and responsive genes, suggesting that the local ethylene production at an early time point after root-cell emergence contributes to the rapid induction of the ethylene-dependent aerenchyma formation in rice. DATA AVAILABILITY: All data included in this study are available upon request by contact with the corresponding author.

Mechanisms of lysigenous aerenchyma formation under abiotic stress.

Lysigenous aerenchyma is a gas space created by cortical cell death that enables efficient oxygen diffusion within plants and reduces the energy costs associated with root cells. Recent studies have demonstrated the benefits of aerenchyma formation under flooding, drought, and nutrient deficiency, although further studies will be necessary to understand the mechanisms involved.

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.

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.

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.

A Role for Auxin in Ethylene-Dependent Inducible Aerenchyma Formation in Rice Roots.

Internal oxygen diffusion from shoot to root tips is enhanced by the formation of aerenchyma (gas space) in waterlogged soils. Lysigenous aerenchyma is created by programmed cell death and subsequent lysis of the root cortical cells. Rice (Oryza sativa) forms aerenchyma constitutively under aerobic conditions and increases its formation under oxygen-deficient conditions. Recently, we have demonstrated that constitutive aerenchyma formation is regulated by auxin signaling mediated by Auxin/indole-3-acetic acid protein (AUX/IAA; IAA). While ethylene is involved in inducible aerenchyma formation, the relationship of auxin and ethylene during aerenchyma formation remains unclear. Here, we examined the effects of oxygen deficiency and ethylene on aerenchyma formation in the roots of a rice mutant (iaa13) in which auxin signaling is suppressed by a mutation in the degradation domain of IAA13 protein. The results showed that AUX/IAA-mediated auxin signaling contributes to ethylene-dependent inducible aerenchyma formation in rice roots. An auxin transport inhibitor abolished aerenchyma formation under oxygen-deficient conditions and reduced the expression of genes encoding ethylene biosynthesis enzymes, further supporting the idea that auxin is involved in ethylene-dependent inducible aerenchyma formation. Based on these studies, we propose a mechanism that underlies the relationship between auxin and ethylene during inducible aerenchyma formation in rice roots.

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.

Root Cortex Provides a Venue for Gas-Space Formation and Is Essential for Plant Adaptation to Waterlogging.

Lysigenous aerenchyma, which develops by death and subsequent lysis of the cortical cells in roots, is essential for internal long-distance oxygen transport from shoot base to root tips of plants in waterlogged soil. Although many studies focus on the amounts of aerenchyma in roots, significance of the size of the root cortex in which aerenchyma forms has received less research attention. In the present study, we evaluated the cross-sectional area of each root tissue in adventitious roots of upland crops, wheat (Triticum aestivum) and maize (Zea mays ssp. mays), and the wetland crop, rice (Oryza sativa) under aerated or stagnant deoxygenated conditions; the latter can mimic the changes in gas composition in waterlogged soils. Our analyses revealed that the areas of whole root and cortex of the three species increased under stagnant conditions. In rice roots, cortex to stele ratio (CSR) and aerenchyma to cortex ratio (ACR), which is associated with the areas of gas spaces, were much higher than those in wheat and maize roots, suggesting that these anatomical features are essential for a high capacity for oxygen transport along roots. To test this hypothesis, rates of radial oxygen loss (ROL), which is the diffusive flux of oxygen from within a root to the external medium, from thick and thin adventitious roots of rice were measured using a cylindrical (root-sleeving) oxygen electrode, for plants with shoots in air and roots in an oxygen-free medium. As expected, the rate of ROL from thick roots, which have larger cortex and aerenchyma areas, was higher than that of thin roots. The rate of ROL was highest at the apical part of rice roots, where aerenchyma was hardly detected, but at which cuboidal cell arrangement in the cortex provides tissue porosity. We conclude that high CSR in combination with large root diameter is a feature which promotes oxygen transport from shoot base to root tips of plants. Moreover, we propose that CSR should be a useful quantitative index for the evaluation and improvement of root traits contributing to tolerance of crops to soil waterlogging.

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.

METALLOTHIONEIN genes encoding ROS scavenging enzymes are down-regulated in the root cortex during inducible aerenchyma formation in rice.

Under waterlogged conditions, roots of gramineous plants form lysigenous aerenchyma (internal gas spaces) by inducing the death of cortical cells. Rice (Oryza sativa) roots induce aerenchyma formation through ethylene- and reactive oxygen species (ROS)-mediated signaling. Metallothionein (MT) is a small, cysteine-rich protein that acts as a ROS scavenger. In rice roots, the expression of MT1a, MT1b, MT1c and MT1Ld were higher than those of the other MT genes. In the root cortex, where aerenchyma forms exclusively, the expression of MT1a, MT1b and MT1Ld was reduced prior to aerenchyma formation. These findings suggest that ROS accumulation in the cortex, which is aided by downregulation of MT1 genes, is needed for aerenchyma formation in rice roots.

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.

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.

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.