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Oryza sativa (rice) plays an essential food security role for more than half of the world's population. Obtaining crops with high levels of disease resistance is a major challenge for breeders, especially today, given the urgent need for agriculture to be more sustainable. Plant resistance genes are mainly encoded by three large leucine-rich repeat (LRR)-containing receptor (LRR-CR) families: the LRR-receptor-like kinase (LRR-RLK), LRR-receptor-like protein (LRR-RLP) and nucleotide-binding LRR receptor (NLR). Using lrrprofiler, a pipeline that we developed to annotate and classify these proteins, we compared three publicly available annotations of the rice Nipponbare reference genome. The extended discrepancies that we observed for LRR-CR gene models led us to perform an in-depth manual curation of their annotations while paying special attention to nonsense mutations. We then transferred this manually curated annotation to Kitaake, a cultivar that is closely related to Nipponbare, using an optimized strategy. Here, we discuss the breakthrough achieved by manual curation when comparing genomes and, in addition to 'functional' and 'structural' annotations, we propose that the community adopts this approach, which we call 'comprehensive' annotation. The resulting data are crucial for further studies on the natural variability and evolution of LRR-CR genes in order to promote their use in breeding future resilient varieties.

Oryza sativa (rice) plays an essential food security role for more than half of the world's population. Obtaining crops with high levels of disease resistance is a major challenge for breeders, especially today, given the urgent need for agriculture to be more sustainable. Plant resistance genes are mainly encoded by three large leucine-rich repeat (LRR)-containing receptor (LRR-CR) families: the LRR-receptor-like kinase (LRR-RLK), LRR-receptor-like protein (LRR-RLP) and nucleotide-binding LRR receptor (NLR). Using lrrprofiler, a pipeline that we developed to annotate and classify these proteins, we compared three publicly available annotations of the rice Nipponbare reference genome. The extended discrepancies that we observed for LRR-CR gene models led us to perform an in-depth manual curation of their annotations while paying special attention to nonsense mutations. We then transferred this manually curated annotation to Kitaake, a cultivar that is closely related to Nipponbare, using an optimized strategy. Here, we discuss the breakthrough achieved by manual curation when comparing genomes and, in addition to 'functional' and 'structural' annotations, we propose that the community adopts this approach, which we call 'comprehensive' annotation. The resulting data are crucial for further studies on the natural variability and evolution of LRR-CR genes in order to promote their use in breeding future resilient varieties.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Background: In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. Results: We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. Conclusion: The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECA

Because of their high level of diversity and complex evolutionary histories, most studies on plant receptor-like kinase subfamilies have focused on their kinase domains. With the large amount of genome sequence data available today, particularly on basal land plants and Charophyta, more attention should be paid to primary events that shaped the diversity of the RLK gene family. We thus focus on the motifs and domains found in association with kinase domains to illustrate their origin, organization, and evolutionary dynamics. We discuss when these different domain associations first occurred and how they evolved, based on a literature review complemented by some of our unpublished results.

Root meristem activity is the most critical process influencing root development. Although several factors that regulate meristem activity have been identified in rice, studies on the enhancement of meristem activity in roots are limited. We identified a T-DNA activation tagging line of a zinc-finger homeobox gene, OsZHD2, which has longer seminal and lateral roots due to increased meristem activity. The phenotypes were confirmed in transgenic plants overexpressing OsZHD2. In addition, the overexpressing plants showed enhanced grain yield under low nutrient and paddy field conditions. OsZHD2 was preferentially expressed in the shoot apical meristem and root tips. Transcriptome analyses and quantitative real-time PCR experiments on roots from the activation tagging line and the wild type showed that genes for ethylene biosynthesis were up-regulated in the activation line. Ethylene levels were higher in the activation lines compared with the wild type. ChIP assay results suggested that OsZHD2 induces ethylene biosynthesis by controlling ACS5 directly. Treatment with ACC (1-aminocyclopropane-1-carboxylic acid), an ethylene precursor, induced the expression of the DR5 reporter at the root tip and stele, whereas treatment with an ethylene biosynthesis inhibitor, AVG (aminoethoxyvinylglycine), decreased that expression in both the wild type and the OsZHD2 overexpression line. These observations suggest that OsZHD2 enhances root meristem activity by influencing ethylene biosynthesis and, in turn, auxin.

Background: In rice, the cortex and outer tissues play a key role in submergence tolerance. The cortex differentiates into aerenchyma, which are air-containing cavities that allow the flow of oxygen from shoots to roots, whereas exodermis suberification and sclerenchyma lignification limit oxygen loss from the mature parts of roots by forming a barrier to root oxygen loss (ROL). The genes and their networks involved in the cellular identity and differentiation of these tissues remain poorly understood. Identification and characterization of key regulators of aerenchyma and ROL barrier formation require determination of the specific expression profiles of these tissues. Results: We optimized an approach combining laser microdissection (LM) and droplet digital RT-PCR (ddRT-PCR) for high-throughput identification of tissue-specific expression profiles. The developed protocol enables rapid (within 3 days) extraction of high-quality RNA from root tissues with a low contamination rate. We also demonstrated the possibility of extracting RNAs from paraffin blocks stored at 4 °C without any loss of quality. We included a detailed troubleshooting guide that should allow future users to adapt the proposed protocol to other tissues and/or species. We demonstrated that our protocol, which combines LM with ddRT-PCR, can be used as a complementary tool to in situ hybridization for tissue-specific characterization of gene expression even with a low RNA concentration input. We illustrated the efficiency of the proposed approach by validating three of four potential tissue-specific candidate genes detailed in the RiceXpro database. Conclusion: The detailed protocol and the critical steps required to optimize its use for other species will democratize tissue-specific transcriptome approaches combining LM with ddRT-PCR for analyses of plants.

Because of their high level of diversity and complex evolutionary histories, most studies on plant receptor-like kinase subfamilies have focused on their kinase domains. With the large amount of genome sequence data available today, particularly on basal land plants and Charophyta, more attention should be paid to primary events that shaped the diversity of the RLK gene family. We thus focus on the motifs and domains found in association with kinase domains to illustrate their origin, organization, and evolutionary dynamics. We discuss when these different domain associations first occurred and how they evolved, based on a literature review complemented by some of our unpublished results.

Genome editing tools have greatly facilitated the functional analysis of genes of interest by targeted mutagenesis. Many usable genome editing tools, including different site-specific nucleases and editor databases that allow single-nucleotide polymorphisms (SNPs) to be introduced at a given site, are now available. These tools can be used to generate high allelic diversity at a given locus to facilitate gene function studies, including examining the role of a specific protein domain or a single amino acid. We compared the effects, efficiencies and mutation types generated by our LbCPF1, SpCAS9 and base editor (BECAS9) constructs for the OsCAO1 gene. SpCAS9 and LbCPF1 have similar efficiencies in generating mutations but differ in the types of mutations induced, with the majority of changes being single-nucleotide insertions and short deletions for SpCAS9 and LbCPF1, respectively. The proportions of heterozygotes also differed, representing a majority in our LbCPF1, while with SpCAS9, we obtained a large number of biallelic mutants. Finally, we demonstrated that it is possible to specifically introduce stop codons using the BECAS9 with an acceptable efficiency of approximately 20%. Based on these results, a rational choice among these three alternatives may be made depending on the type of mutation that one wishes to introduce, the three systems being complementary. SpCAS9 remains the best choice to generate KO mutations in primary transformants, while if the desired gene mutation interferes with regeneration or viability, the use of our LbCPF1 construction will be preferred, because it produces mainly heterozygotes. LbCPF1 has been described in other studies as being as effective as SpCAS9 in generating homozygous and biallelic mutations. It will remain to be clarified in the future, whether the different LbCFP1 constructions have different efficiencies and determine the origin of these differences. Finally, if one wishes to specifically introduce stop codons, BECA

In the last 15 years, outstanding progress has been made in understanding the function of meiotic genes in the model dicot and monocot plants Arabidopsis and rice (Oryza sativa L.), respectively. This knowledge allowed to modulate meiotic recombination in Arabidopsis and, more recently, in rice. For instance, the overall frequency of crossovers (COs) has been stimulated 2.3‐ and 3.2‐fold through the inactivation of the rice FANCM and RECQ4 DNA helicases, respectively, two genes involved in the repair of DNA double‐strand breaks (DSBs) as noncrossovers (NCOs) of the Class II crossover pathway. Differently, the programmed induction of DSBs and COs at desired sites is currently explored by guiding the SPO11‐1 topoisomerase‐like transesterase, initiating meiotic recombination in all eukaryotes, to specific target regions of the rice genome. Furthermore, the inactivation of 3 meiosis‐specific genes, namely PAIR1, OsREC8 and OsOSD1, in the Mitosis instead of Meiosis (MiMe) mutant turned rice meiosis into mitosis, thereby abolishing recombination and achieving the first component of apomixis, apomeiosis. The successful translation of Arabidopsis results into a crop further allowed the implementation of two breakthrough strategies that triggered parthenogenesis from the MiMe unreduced clonal egg cell and completed the second component of diplosporous apomixis. Here, we review the most recent advances in and future prospects of the manipulation of meiotic recombination in rice and potentially other major crops, all essential for global food security.

In the last 15 years, outstanding progress has been made in understanding the function of meiotic genes in the model dicot and monocot plants Arabidopsis and rice (Oryza sativa L.), respectively. This knowledge allowed to modulate meiotic recombination in Arabidopsis and, more recently, in rice. For instance, the overall frequency of crossovers (COs) has been stimulated 2.3‐ and 3.2‐fold through the inactivation of the rice FANCM and RECQ4 DNA helicases, respectively, two genes involved in the repair of DNA double‐strand breaks (DSBs) as noncrossovers (NCOs) of the Class II crossover pathway. Differently, the programmed induction of DSBs and COs at desired sites is currently explored by guiding the SPO11‐1 topoisomerase‐like transesterase, initiating meiotic recombination in all eukaryotes, to specific target regions of the rice genome. Furthermore, the inactivation of 3 meiosis‐specific genes, namely PAIR1, OsREC8 and OsOSD1, in the Mitosis instead of Meiosis (MiMe) mutant turned rice meiosis into mitosis, thereby abolishing recombination and achieving the first component of apomixis, apomeiosis. The successful translation of Arabidopsis results into a crop further allowed the implementation of two breakthrough strategies that triggered parthenogenesis from the MiMe unreduced clonal egg cell and completed the second component of diplosporous apomixis. Here, we review the most recent advances in and future prospects of the manipulation of meiotic recombination in rice and potentially other major crops, all essential for global food security.

Salinity tolerance is an important quality for European rice grown in river deltas. We evaluated the salinity tolerance of a panel of 235 temperate japonica rice accessions genotyped with 30,000 SNP markers. The panel was exposed to mild salt stress (50 mM NaCl; conductivity of 6 dS m-1) at the seedling stage. Eight different root and shoot growth parameters were measured for both the control and stressed treatments. The Na+ and K+ mass fractions of the stressed plants were measured using atomic absorption spectroscopy. The salt treatment affected plant growth, particularly the shoot parameters. The panel showed a wide range of Na+/K+ ratio and the temperate accessions were distributed over an increasing axis, from the most resistant to the most susceptible checks. We conducted a genome-wide association study on indices of stress response and ion mass fractions in the leaves using a classical mixed model controlling structure and kinship. A total of 27 QTLs validated by sub-sampling were identified. For indices of stress responses, we also used another model that focused on marker × treatment interactions and detected 50 QTLs, three of which were also identified using the classical method. We compared the positions of the significant QTLs to those of approximately 300 genes that play a role in rice salt tolerance. The positions of several QTLs were close to those of genes involved in calcium signaling and metabolism, while other QTLs were close to those of kinases. These results reveal the salinity tolerance of accessions with a temperate japonica background. Although the detected QTLs must be confirmed by other approaches, the number of associations linked to candidate genes involved in calcium-mediated ion homeostasis highlights pathways to explore in priority to understand the salinity tolerance of temperate rice.

Background: The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in A. thaliana. However, such an approach is relatively ineffective in species with more complex and thicker root systems. Results: We adapted a PI counter-staining protocol to visualize the internal 3D architecture of rice root meristems using multiphoton microscopy. This protocol is simple and compatible with the main fluorophores (CFP, GFP and mCherry). The efficiency and applicability of this protocol were demonstrated by screening a population of 57 enhancer trap lines. We successfully characterized GFP expression in all of the lines and identified 5 lines with tissue-specific expression. Conclusions: All of these resources are now available for the rice community and represent critical tools for future studies of root development.

Root traits such as root angle and hair length influence resource acquisition particularly for immobile nutrients like phosphorus (P). Here, we attempted to modify root angle in rice by disrupting the OsAUX1 auxin influx transporter gene in an effort to improve rice P acquisition efficiency. We show by X-ray microCT imaging that root angle is altered in the osaux1 mutant, causing preferential foraging in the top soil where P normally accumulates, yet surprisingly, P acquisition efficiency does not improve. Through closer investigation, we reveal that OsAUX1 also promotes root hair elongation in response to P limitation. Reporter studies reveal that auxin response increases in the root hair zone in low P environments. We demonstrate that OsAUX1 functions to mobilize auxin from the root apex to the differentiation zone where this signal promotes hair elongation when roots encounter low external P. We conclude that auxin and OsAUX1 play key roles in promoting root foraging for P in rice.

Background: The clear visualization of 3D organization at the cellular level in plant tissues is needed to fully understand plant development processes. Imaging tools allow the visualization of the main fluorophores and in vivo growth monitoring. Confocal microscopy coupled with the use of propidium iodide (PI) counter-staining is one of the most popular tools used to characterize the structure of root meristems in A. thaliana. However, such an approach is relatively ineffective in species with more complex and thicker root systems. Results: We adapted a PI counter-staining protocol to visualize the internal 3D architecture of rice root meristems using multiphoton microscopy. This protocol is simple and compatible with the main fluorophores (CFP, GFP and mCherry). The efficiency and applicability of this protocol were demonstrated by screening a population of 57 enhancer trap lines. We successfully characterized GFP expression in all of the lines and identified 5 lines with tissue-specific expression. Conclusions: All of these resources are now available for the rice community and represent critical tools for future studies of root development.

Root traits such as root angle and hair length influence resource acquisition particularly for immobile nutrients like phosphorus (P). Here, we attempted to modify root angle in rice by disrupting the OsAUX1 auxin influx transporter gene in an effort to improve rice P acquisition efficiency. We show by X-ray microCT imaging that root angle is altered in the osaux1 mutant, causing preferential foraging in the top soil where P normally accumulates, yet surprisingly, P acquisition efficiency does not improve. Through closer investigation, we reveal that OsAUX1 also promotes root hair elongation in response to P limitation. Reporter studies reveal that auxin response increases in the root hair zone in low P environments. We demonstrate that OsAUX1 functions to mobilize auxin from the root apex to the differentiation zone where this signal promotes hair elongation when roots encounter low external P. We conclude that auxin and OsAUX1 play key roles in promoting root foraging for P in rice.

Background: Cell biology approach using membrane protein markers tagged with fluorescent proteins highlights the dynamic behaviour of plant cell membranes, not only in the standard but also in changing environmental conditions. In the past, this strategy has been extensively developed in plant models such as Arabidopsis. Results: Here, we generated a set of transgenic lines expressing membrane protein markers to extend this approach to rice, one of the most cultivated crop in the world and an emerging plant model. Lines expressing individually eight membrane protein markers including five aquaporins (OsPIP1;1, OsPIP2;4, OsPIP2;5, OsTIP1;1, OsTIP2;2) and three endosomal trafficking proteins (OsRab5a, OsGAP1, OsSCAMP1) were obtained. Importantly, we challenged in roots the aquaporin-expressing transgenic lines upon salt and osmotic stress and uncovered a highly dynamic behaviour of cell membrane. Conclusion: We have uncovered the relocalization and dynamics of plasma membrane aquaporins upon salt and osmotic stresses in rice. Importantly, our data support a model where relocalization of OsPIPs is concomitant with their high cycling dynamics.

Salinity tolerance is an important quality for European rice grown in river deltas. We evaluated the salinity tolerance of a panel of 235 temperate japonica rice accessions genotyped with 30,000 SNP markers. The panel was exposed to mild salt stress (50 mM NaCl; conductivity of 6 dS m-1) at the seedling stage. Eight different root and shoot growth parameters were measured for both the control and stressed treatments. The Na+ and K+ mass fractions of the stressed plants were measured using atomic absorption spectroscopy. The salt treatment affected plant growth, particularly the shoot parameters. The panel showed a wide range of Na+/K+ ratio and the temperate accessions were distributed over an increasing axis, from the most resistant to the most susceptible checks. We conducted a genome-wide association study on indices of stress response and ion mass fractions in the leaves using a classical mixed model controlling structure and kinship. A total of 27 QTLs validated by sub-sampling were identified. For indices of stress responses, we also used another model that focused on marker × treatment interactions and detected 50 QTLs, three of which were also identified using the classical method. We compared the positions of the significant QTLs to those of approximately 300 genes that play a role in rice salt tolerance. The positions of several QTLs were close to those of genes involved in calcium signaling and metabolism, while other QTLs were close to those of kinases. These results reveal the salinity tolerance of accessions with a temperate japonica background. Although the detected QTLs must be confirmed by other approaches, the number of associations linked to candidate genes involved in calcium-mediated ion homeostasis highlights pathways to explore in priority to understand the salinity tolerance of temperate rice.

Background: The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. Findings: We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. Conclusions: All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

Leucine-Rich Repeats Receptor-Like Kinase (LRR-RLK) genes represent a large and complex gene family in plants, mainly involved in development and stress responses. These receptors are composed of an LRR-containing extracellular domain (ECD), a transmembrane domain (TM) and an intracellular kinase domain (KD). To provide new perspectives on functional analyses of these genes in model and non-model plant species, we performed a phylogenetic analysis on 8,360 LRR-RLK receptors in 31 angiosperm genomes (8 monocots and 23 dicots). We identified 101 orthologous groups (OGs) of genes being conserved among almost all monocot and dicot species analyzed. We observed that more than 10% of these OGs are absent in the Brassicaceae species studied. We show that the ECD structural features are not always conserved among orthologs, suggesting that functions may have diverged in some OG sets. Moreover, we looked at targets of positive selection footprints in 12 pairs of OGs and noticed that depending on the subgroups, positive selection occurred more frequently either in the ECDs or in the KDs.

Background: The DEFECTIVE IN OUTER CELL LAYER SPECIFICATION 1 (DOCS1) gene belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) subfamily. It has been discovered few years ago in Oryza sativa (rice) in a screen to isolate mutants with defects in sensitivity to aluminum. The c68 (docs1–1) mutant possessed a nonsense mutation in the C-terminal part of the DOCS1 kinase domain. Findings: We have generated a new loss-of-function mutation in the DOCS1 gene (docs1–2) using the CRISPR-Cas9 technology. This new loss-of-function mutant and docs1–1 present similar phenotypes suggesting the original docs1–1 was a null allele. Besides the aluminum sensitivity phenotype, both docs1 mutants shared also several root phenotypes described previously: less root hairs and mixed identities of the outer cell layers. Moreover, our new results suggest that DOCS1 could also play a role in root cap development. We hypothesized these docs1 root phenotypes may affect gravity responses. As expected, in seedlings, the early gravitropic response was delayed. Furthermore, at adult stage, the root gravitropic set angle of docs1 mutants was also affected since docs1 mutant plants displayed larger root cone angles. Conclusions: All these observations add new insights into the DOCS1 gene function in gravitropic responses at several stages of plant development.

Leucine-Rich Repeats Receptor-Like Kinase (LRR-RLK) genes represent a large and complex gene family in plants, mainly involved in development and stress responses. These receptors are composed of an LRR-containing extracellular domain (ECD), a transmembrane domain (TM) and an intracellular kinase domain (KD). To provide new perspectives on functional analyses of these genes in model and non-model plant species, we performed a phylogenetic analysis on 8,360 LRR-RLK receptors in 31 angiosperm genomes (8 monocots and 23 dicots). We identified 101 orthologous groups (OGs) of genes being conserved among almost all monocot and dicot species analyzed. We observed that more than 10% of these OGs are absent in the Brassicaceae species studied. We show that the ECD structural features are not always conserved among orthologs, suggesting that functions may have diverged in some OG sets. Moreover, we looked at targets of positive selection footprints in 12 pairs of OGs and noticed that depending on the subgroups, positive selection occurred more frequently either in the ECDs or in the KDs.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despitebeing present at low concentrations in contaminated soils (belowlM), cesium (Cs+) can be taken up bycrops and transported to their edible parts. This plant capacity to take up Cs+from low concentrations hasnotably affected the production of rice (Oryza sativaL.) in Japan after the nuclear accident at Fukushima in2011. Several strategies have been put into practice to reduce Cs+content in this crop species such as con-taminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management,with limited impact or pernicious side-effects. Conversely, the development of biotechnological approachesaimed at reducing Cs+accumulation in rice remain challenging. Here, we show that inactivation of the Cs+-permeable K+transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+uptake by riceplants. Cs+uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similarkinetics parameters. In rice, Cs+uptake is dependent on two functional properties of OsHAK1: (i) a poorcapacity of this system to discriminate between Cs+and K+; and (ii) a high capacity to transport Cs+fromvery low external concentrations that is likely to involve an active transport mechanism. In an experimentwith a Fukushima soil highly contaminated with137Cs+, plants lacking OsHAK1 function displayed strikinglyreduced levels of137Cs+in roots and shoots. These results open stimulating perspectives to smartly producesafe food in regions contaminated by nuclear accidents

The formation and differentiation of aerenchyma, i.e., air-containing cavities that are critical for flooding tolerance, take place exclusively in the cortex. The understanding of development and differentiation of the cortex is thus an important issue; however, studies on this tissue are limited, partly because of the lack of available molecular tools. We screened a commercially available library of cell wall antibodies to identify markers of cortical tissue in rice roots. Out of the 174 antibodies screened, eight were cortex-specific. Our analysis revealed that two types of cortical tissues are present in rice root seedlings. We named these cell layers “inner” and “outer” based on their location relative to the stele. We then used the antibodies to clarify cell identity in lateral roots. Without these markers, previous studies could not distinguish between the cortex and sclerenchyma in small lateral roots. By immunostaining lateral root sections, we showed that the internal ground tissue in small lateral roots has outer cortical identity.

Plants are constantly exposed to a variety of biotic and abiotic stresses that reduce their fitness and performance. At the molecular level, the perception of extracellular stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades, in which protein phosphorylation plays a central role. Several studies have shown that some members of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family are involved in stress and developmental pathways. We report here a systematic analysis of the role of the members of this gene family by mutant phenotyping in the monocotyledon model plant rice, Oryza sativa. We have then targeted 176 of the ∼320 LRR-RLK genes (55.7%) and genotyped 288 mutant lines. Position of the insertion was confirmed in 128 lines corresponding to 100 LRR-RLK genes (31.6% of the entire family). All mutant lines harboring homozygous insertions have been screened for phenotypes under normal conditions and under various abiotic stresses. Mutant plants have been observed at several stages of growth, from seedlings in Petri dishes to flowering and grain filling under greenhouse conditions. Our results show that 37 of the LRR-RLK rice genes are potential targets for improvement especially in the generation of abiotic stress tolerant cereals.

The formation and differentiation of aerenchyma, i.e., air-containing cavities that are critical for flooding tolerance, take place exclusively in the cortex. The understanding of development and differentiation of the cortex is thus an important issue; however, studies on this tissue are limited, partly because of the lack of available molecular tools. We screened a commercially available library of cell wall antibodies to identify markers of cortical tissue in rice roots. Out of the 174 antibodies screened, eight were cortex-specific. Our analysis revealed that two types of cortical tissues are present in rice root seedlings. We named these cell layers “inner” and “outer” based on their location relative to the stele. We then used the antibodies to clarify cell identity in lateral roots. Without these markers, previous studies could not distinguish between the cortex and sclerenchyma in small lateral roots. By immunostaining lateral root sections, we showed that the internal ground tissue in small lateral roots has outer cortical identity.

Plants are constantly exposed to a variety of biotic and abiotic stresses that reduce their fitness and performance. At the molecular level, the perception of extracellular stimuli and the subsequent activation of defense responses require a complex interplay of signaling cascades, in which protein phosphorylation plays a central role. Several studies have shown that some members of the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family are involved in stress and developmental pathways. We report here a systematic analysis of the role of the members of this gene family by mutant phenotyping in the monocotyledon model plant rice, Oryza sativa. We have then targeted 176 of the ∼320 LRR-RLK genes (55.7%) and genotyped 288 mutant lines. Position of the insertion was confirmed in 128 lines corresponding to 100 LRR-RLK genes (31.6% of the entire family). All mutant lines harboring homozygous insertions have been screened for phenotypes under normal conditions and under various abiotic stresses. Mutant plants have been observed at several stages of growth, from seedlings in Petri dishes to flowering and grain filling under greenhouse conditions. Our results show that 37 of the LRR-RLK rice genes are potential targets for improvement especially in the generation of abiotic stress tolerant cereals.

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signalling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxinmaxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture.

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signalling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxinmaxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture.

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signalling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxinmaxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture.

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signalling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxinmaxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture.

The hormone auxin is critical for many plant developmental processes. Unlike the model eudicot plant Arabidopsis (Arabidopsis thaliana), auxin distribution and signaling in rice tissues has not been systematically investigated due to the absence of suitable auxin response reporters. In this study we observed the conservation of auxin signalling components between Arabidopsis and model monocot crop rice (Oryza sativa), and generated complementary types of auxin biosensor constructs, one derived from the Aux/IAA-based biosensor DII-VENUS but constitutively driven by maize ubiquitin-1 promoter, and the other termed DR5-VENUS in which a synthetic auxin-responsive promoter (DR5rev) was used to drive expression of the yellow fluorescent protein (YFP). Using the obtained transgenic lines, we observed that during the vegetative development, accumulation of DR5-VENUS signal was at young and mature leaves, tiller buds and stem base. Notably, abundant DR5-VENUS signals were observed in the cytoplasm of cortex cells surrounding lateral root primordia (LRP) in rice. In addition, auxinmaxima and dynamic re-localization were seen at the initiation sites of inflorescence and spikelet primordia including branch meristems (BMs), female and male organs. The comparison of these observations among Arabidopsis, rice and maize suggests the unique role of auxin in regulating rice lateral root emergence and reproduction. Moreover, protein localization of auxin transporters PIN1 homologs and GFP tagged OsAUX1 overlapped with DR5-VENUS during spikelet development, helping validate these auxin response reporters are reliable markers in rice. This work firstly reveals the direct correspondence between auxin distribution and rice reproductive and root development at tissue and cellular level, and provides high-resolution auxin tools to probe fundamental developmental processes in rice and to establish links between auxin, development and agronomical traits like yield or root architecture.