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The nutritional enhancement of potato plants (Solanum tuberosum L.,) is highly critical. As it is considered a worldwide basic vegetarian nutrition to maintain health. S. tuberosum is one of the foremost staples and the world's fourth-largest food crop. In advance, its need is increasing because of its high-industrial value and population blast. To improve both potato growth and behavior under harsh environmental conditions, we produced transgenic potato plants overexpressing either VvNHX (a sodium proton antiporter from Vitis vinifera), VvCLC (a chloride channel from Vitis vinifera), or both. Control and transgenic plants were grown in greenhouse and field under non-stressed conditions for 85 days in order to characterize their phenotype and evaluate their agronomical performance. To this aim, the evaluation of plant growth parameters, tuber yields and characteristics (calibers, eye number and color), the chemical composition of tubers, was conducted and compared between the different lines. The obtained results showed that transgenic plants displayed an improved growth (flowering precocity, gain of vigor and better vegetative growth) along with enhanced tuber yields and quality (increased protein and starch contents). Our findings provide then insight into the role played by the VvNHX antiport and the VvCLC channel and a greater understanding of the effect of their overexpression in potato plants.

K+ uptake to root conducting cells is a prerequisite for its release to the xylem vessels and long-distance delivery to the aerial parts, though the molecular mechanism underpinning such process remains insufficiently understood. Here we report the discovery in rice root of an essential component involved in loading of K+ to the stelar tissues encoded by an unusual short Shaker K+ channel OsK2.2. OsK2.2 represents a novel type of short Shaker channels lacking the sequences immediately downstream of the putative cyclic nucleotide binding domain that present to the C-termini of all plant Shaker channels reported to date. In silico analysis suggests that such short Shaker channels are monocot-specific and especially found in the Poaceae species. Functional assessment in Xenopus oocytes reveals that OsK2.2 uses such innate C-terminal shortness as an intrinsic strategy for maintaining large capacity of K+ uptake to the cells. OsK2.2 predominantly localizes to the phloem and xylem peripheral cells of rice root vasculature. Disruption of OsK2.2 function causes approximately 30% lower K+ in both phloem and xylem sap and reduces K+ accumulation to the shoots. OsK2.2 strongly contributes to the grain yield of rice under field conditions. The essential contribution of such particular type of inward Shaker channel, operating in stelar tissues, to K+ transport between roots and shoots and to grain yield, here evidenced in rice, may be a common trait amongst Poaceae species.

In Arabidopsis, chromosomal double-strand breaks at meiosis are presumably catalyzed by two distinct SPO11 transesterases, AtSPO11-1 and AtSPO11-2, together with M-TOPVIB. To clarify the roles of the SPO11 paralogs in rice, we used CRISPR/Cas9 mutagenesis to produce null biallelic mutants in OsSPO11-1, OsSPO11-2, and OsSPO11-4. Similar to Osspo11-1, biallelic mutations in the first exon of OsSPO11-2 led to complete panicle sterility. Conversely, all Osspo11-4 biallelic mutants were fertile. To generate segregating Osspo11-2 mutant lines, we developed a strategy based on dual intron targeting. Similar to Osspo11-1, the pollen mother cells of Osspo11-2 progeny plants showed an absence of bivalent formation at metaphase I, aberrant segregation of homologous chromosomes, and formation of non-viable tetrads. In contrast, the chromosome behavior in Osspo11-4 male meiocytes was indistinguishable from that in the wild type. While similar numbers of OsDMC1 foci were revealed by immunostaining in wild-type and Osspo11-4 prophase pollen mother cells (114 and 101, respectively), a surprisingly high number (85) of foci was observed in the sterile Osspo11-2 mutant, indicative of a divergent function between OsSPO11-1 and OsSPO11-2. This study demonstrates that whereas OsSPO11-1 and OsSPO11-2 are the likely orthologs of AtSPO11-1 and AtSPO11-2, OsSPO11-4 has no major role in wild-type rice meiosis.

Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.

20 páginas.- 9 figuras.- 1 tablas.- referencias.- Supplemental Data - Supplemental Figures 1-9 y Supplemental Table 1-5.- Freely accesible article at https://doi.org/10.1104/pp.19.00324 .- Freely accesible article at https://www.ncbi.nlm.nih.gov/pubmed/30992336, Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt-sensitive and the undue accumulation of sodium ions (Na+) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na+/H+ exchanger protein SOS1 is the sole Na+ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na+ flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na+ intake and impaired Na+ loading into the xylem, thus indicating that SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots. The acute Na+ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast to that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1. In summary, the plasma membrane Na+/H+ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na+ homeostasis and possibly contributing to the sensing of sodicity stress., This work was supported by grants BIO2015-70946-R to FJQ, and grants BFU2015 64671-R and BIO2016-81957-REDT to JMP from MINECO (co-financed by the European Regional Development Fund), and by the Rural Development Administration, Republic of Korea (SSAC grants PJ01108101 to D-JY and PJ01318205 to JMP). Part of this work was conducted at the Rice Functional Genomics Platform (REFUGE), Montpellier France, supported by an Agropolis Fondation grant to DM and EG. FGA was supported by a 'Juan de la Cierva' aid from MINECO.

International audience; The AlSAP gene, which has been isolated from the C4 halophyte grass Aeluropus littoralis, a member of the stress-associated protein gene family. Like other members of this family, AlSAP contains A20 and AN1 zinc-finger domains, and its transcription is induced by abiotic stresses such as salinity, drought, and temperature as well as by several hormones, including abscisic and salicylic acids. Additionally, AlSAP overexpression in rice has been demonstrated to enhance cold and drought stress tolerance. In the present study, we investigated the transcriptional deregulation of stress-related genes following ectopic expression of the AlSAP gene in two transgenic rice lines, namely, RN4 and RN5. Compared with control plants, a total of 1734 and 3650 annotated genes were found to be differentially expressed in the leaves and roots of the RN4 and RN5 lines, respectively. AlSAP transcript accumulation primed the expression of abiotic-stress-related genes involved in transcription, signaling, protein degradation, and hormonal homeostasis in rice plants grown under unstressed conditions. We hypothesized that this priming underlies the enhanced tolerance to abiotic stresses previously reported in AlSAP rice lines. Notably, AlSAP also induces transcript accumulation of genes known to be major negative regulators of pathogen response in rice. Consistently, our results showed that abiotic stress-tolerant AlSAP rice lines also exhibit enhanced susceptibility to rice blast fungus Magnaporthe oryzae.

International audience; Genome editing technologies, mainly CRISPR/CAS9, are revolutionizing plant biology and breeding. Since the demonstration of its effectiveness in eukaryotic cells, a very large number of derived technologies has emerged. Demonstrating and comparing the effectiveness of all these new technologies in entire plants is a long, tedious, and labor-intensive process that generally involves the production of transgenic plants and their analysis. Protoplasts, plant cells free of their walls, offer a simple, high-throughput system to test the efficiency of these editing technologies in a few weeks' time span. We have developed a routine protocol using protoplasts to test editing technologies in rice. Our protocol allows to test more than 30 constructs in protoplasts prepared from leaf tissues of 100, 9-11-day-old seedlings. CRISPR/CAS9 construct effectiveness can be clearly established within less than a week. We provide here a full protocol, from designing sgRNA to mutation analysis.

International audience; The manipulation of meiotic recombination in crops is essential to develop new plant varieties rapidly, helping to produce more cultivars in a sustainable manner. One option is to control the formation and repair of the meiosis-specific DNA double-strand breaks (DSBs) that initiate recombination between the homologous chromosomes and ultimately lead to crossovers. These DSBs are introduced by the evolutionarily conserved topoisomerase-like protein SPO11 and associated proteins. Here, we characterized the homoeologous copies of the SPO11-1 protein in hexaploid bread wheat (Triticum aestivum). The genome contains three SPO11-1 gene copies that exhibit 93-95% identity at the nucleotide level, and clearly the A and D copies originated from the diploid ancestors Triticum urartu and Aegilops tauschii, respectively. Furthermore, phylogenetic analysis of 105 plant genomes revealed a clear partitioning between monocots and dicots, with the seven main motifs being almost fully conserved, even between clades. The functional similarity of the proteins among monocots was confirmed through complementation analysis of the Oryza sativa (rice) spo11-1 mutant by the wheat TaSPO11-1-5D coding sequence. Also, remarkably, although the wheat and Arabidopsis SPO11-1 proteins share only 55% identity and the partner proteins also differ, the TaSPO11-1-5D cDNA significantly restored the fertility of the Arabidopsis spo11-1 mutant, indicating a robust functional conservation of the SPO11-1 protein activity across distant plants. These successful heterologous complementation assays, using both Arabidopsis and rice hosts, are good surrogates to validate the functionality of candidate genes and cDNA, as well as variant constructs, when the transformation and mutant production in wheat is much longer and more tedious.

International audience; Rice, the staple food for more than half of humankind and the model monocot crop, was the first cereal in which efficient gene transfer procedures were implemented: 30 years ago, the first transgenic rice plant was regenerated following direct gene transfer to cell suspension-derived protoplasts. Shortly thereafter, transgenic plants were regenerated from zygotic embryo-derived cells following their subjection to micro-projectile acceleration and Agrobacterium-mediated transfection treatments. The high efficiency of transfer deoxyribonucleic acid (T-DNA) integration in seed embryo-derived cells of rice has allowed the transfer of genes of agronomical relevance and the generation of large collections of insertion lines and has provided a key contribution to deciphering the function of more than 2000 rice genes. The high efficiency of T-DNA integration in seed embryo-derived cells of rice also permitted the first implementation of gene targeting and knock-in (KI) events, relying on the albeit very low natural frequency of homology-directed repair (HDR) in the rice genome. In the late 2000s, the advent of site-directed nucleases (SDNs) that induce either single or double-strand breaks at a high frequency and their rapid application to rice permitted routine targeted mutagenesis, which can be multiplexed to simultaneously alter several targets or create deletions, and base and gene editing (e.g. correction of amino acids). Currently, the challenge remains to attain a high frequency of KI and replacement of long stretches of DNA for protein domain or coding sequence swapping. We present herein a historical perspective of the advances that have been readily implemented to determine the function of rice genes and to manipulate traits of agronomical relevance. Two main bottlenecks remain to be alleviated in rice genomic engineering: the low frequency of HDR and the genotype dependence of gene transfer efficiency. Alleviation of these bottlenecks is needed to reach the potential of intra- and interspecific gene replacement and SDN-mediated multiplex editing of alleles in elite materials, which will assist in the breeding and deployment of rice cultivars embedded in sustainable and climate-smart agricultural practices.

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1 , a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice ( Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

International audience; There is little known about the function of rice hexokinases (HXKs) in planta. We characterized hxk5-1, a Tos17 mutant of OsHXK5 that is up-regulated in maturing pollen, a stage when starch accumulates. Progeny analysis of self-pollinated heterozygotes of hxk5-1 and reciprocal crosses between the wild-type and heterozygotes revealed that loss of HXK5 causes male sterility. Homozygous hxk5-1, produced via anther culture, and additional homozygous hxk5-2, hxk5-3 and hxk5-4 lines created by CRISPR/Cas9 confirmed the male-sterile phenotype. In vitro pollen germination ability and in vivo pollen tube growth rate were significantly reduced in the hxk5 mutant pollen. Biochemical analysis of anthers with the mutant pollen revealed significantly reduced hexokinase activity and starch content, although they were sufficient to produce some viable seed. However, the mutant pollen was unable to compete successfully against wild-type pollen. Expression of the catalytically inactive OsHXK5-G113D did not rescue the hxk5 male-sterile phenotype, indicating that its catalytic function was responsible for pollen fertility, rather than its role in sugar sensing and signaling. Our results demonstrate that OsHXK5 contributes to a large portion of the hexokinase activity necessary for the starch utilization pathway during pollen germination and tube growth, as well as for starch biosynthesis during pollen maturation.

We characterized a novel stress tolerance gene from Aeluropus littoralis encoding a protein designed as AlSRG1 ( A . l ittoralis S tress- R elated G ene 1). Phylogenetic and conserved domain prediction identified AlSRG1 as an uncharacterized protein of unknown function. The conserved domain database revealed that AlSRG1 contains an RNA-recognition motif (RRM). This domain was highlighted in AlSRG1 primary, secondary and tertiary structures with the two conserved motifs RNP1 and RNP2 being part of it. These two conserved motifs contain the active site for RNA-binding and a 3D-model of conserved domain (AlSRG1p) was built from known X-ray structures and sequence alignments. The analysis with RT-qPCR revealed higher AlSRG1 transcript accumulation under salt and osmotic stresses. Overexpression of AlSRG1 in transgenic tobacco plants improved both salt and osmotic tolerance and was accompanied by higher seedling survival rates, better growth parameters and ROS accumulation than non-transgenic plants (NT). Furthermore, the transgenic tobacco plants were able to complete their life cycle and to produce viable seeds while the NT plants died at the vegetative stage. The AlSRG1 transgenic lines compared to NT tobacco, showed a significantly lower water loss rate in senescent-basal leaves. AlSRG1 overexpression also enhanced the transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results show that AlSRG1 may play an important positive modulation role in abiotic stress tolerance.

Herein, we report isolation of the AlTMP2 gene from the halophytic C4 grass Aeluropus littoralis. The subcellular localization suggested that AlTMP2 is a plasma membrane protein. In A. littoralis exposed to salt and osmotic stresses, the AlTMP2 gene was induced early and at a high rate, but was upregulated relatively later in response to abscisic acid and cold treatments. Expression of AlTMP2 in tobacco conferred improved tolerance against salinity, osmotic, H2O2, heat, and freezing stresses at the germination and seedling stages. Under control conditions, no growth or yield penalty were mentioned in transgenic plants due to the constitutive expression of AlTMP2. Interestingly, under greenhouse conditions, the seed yield of transgenic plants was significantly higher than that of non-transgenic (NT) plants grown under salt or drought stress. Furthermore, AlTMP2 plants had less electrolyte leakage, higher membrane stability, and lower Na+ and higher K+ accumulation than NT plants. Finally, six stress-related genes were shown to be deregulated in AlTMP2 plants relative to NT plants under both control and stress conditions. Collectively, these results indicate that AlTMP2 confers abiotic stress tolerance by improving ion homeostasis and membrane integrity, and by deregulating certain stress-related genes.

The large French research project GENIUS (2012–2019, https://www6.inra.genius-project_eng/ ) provides a good showcase of current genome editing techniques applied to crop plants. It addresses a large variety of agricultural species (rice, wheat, maize, tomato, potato, oilseed rape, poplar, apple and rose) together with some models (Arabidopsis, Brachypodium, Physcomitrella). Using targeted mutagenesis as its work horse, the project is limited to proof of concept under confined conditions. It mainly covers traits linked to crop culture, such as disease resistance to viruses and fungi, flowering time, plant architecture, tolerance to salinity and plant reproduction but also addresses traits improving the quality of agricultural products for industrial purposes. Examples include virus resistant tomato, early flowering apple and low-amylose starch potato. The wide range of traits illustrates the potential of genome editing towards a more sustainable agriculture through the reduction of pesticides and to the emergence of innovative bio-economy sectors based on custom tailored quality traits.

Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na(+)) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na(+)/H(+) exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na(+) efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na(+) flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na(+) intake and impaired Na(+) loading into the xylem, thus indicating that SOS1 controls net root Na(+) uptake and long-distance Na(+) transport to shoots. The acute Na(+) sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1. In summary, the plasma membrane Na(+)/H(+) exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na(+) homeostasis and possibly contributing to the sensing of sodicity stress.

We investigated the expression in transgenic rice of a synthetic gene encoding the toxic part of the Bacillus thuringiensis Cry1Ba endotoxin, which was shown to exhibit a tenfold lower lethal concentration 50 (LC50) than Cry1Ac in a Striped Stem Borer (SSB) diet incorporation assay. The 1.950-bp synthetic cry1B gene, possessing an overall GC content of 58 %, was cloned under the control of the maize ubiquitin promoter first intron and first exon regions. The resulting vector, designated as pUbi-cry1B, was transferred to two commercial Mediterranean cultivars of rice, Ariete and Senia, using microprojectile acceleration-mediated transformation. Thirty-two and 47 T0 events were generated in cvs. Ariete and Senia, respectively. Southern blot and immunoblot analyses allowed the identification of 7 Senia and 1 Ariete events harbouring both an intact gene cassette and expressing Cry1B at a level ranging from 0.01 % to 0.4 % of the total soluble proteins. Three Senia and 1 Ariete events were found to be protected against second instar SSB larvae in whole plant feeding assays, exhibiting 90-100 % mortality 7 days after infestation. Spatial and temporal variation in transgene expression was further examined in resistant event 64 of cv. Ariete. Stable accumulation of Cry1B, representing 0.4 % of the total soluble proteins, was observed over the T2 to T4 generations in leaf tissue 20, 40, 70 and 90 days after germination in both young and old leaves and in internodes. Ariete event 64 was found to be fully protected from attacks of third and fourth instar SSB larvae over subsequent generations.

Background Productivity of important crop rice is greatly affected by salinity. The plant hormone jasmonate plays a vital role in salt stress adaptation, but also evokes detrimental side effects if not timely shut down again. As novel strategy to avoid such side effects, OsJAZ8, a negative regulator of jasmonate signalling, is expressed under control of the salt-inducible promoter of the transcription factor ZOS3–11, to obtain a transient jasmonate signature in response to salt stress. To modulate the time course of jasmonate signalling, either a full-length or a dominant negative C-terminally truncated version of OsJAZ8 driven by the ZOS3–11 promoter were expressed in a stable manner either in tobacco BY-2 cells, or in japonica rice. Results The transgenic tobacco cells showed reduced mortality and efficient cycling under salt stress adaptation. This was accompanied by reduced sensitivity to Methyl jasmonate and increased responsiveness to auxin. In the case of transgenic rice, the steady-state levels of OsJAZ8 transcripts were more efficiently induced under salt stress compared to the wild type, this induction was more pronounced in the dominant-negative OsJAZ8 variant. Conclusions The result concluded that, more efficient activation of OsJAZ8 was accompanied by improved salt tolerance of the transgenic seedlings and demonstrates the impact of temporal signatures of jasmonate signalling for stress tolerance. Electronic supplementary material The online version of this article (10.1186/s12870-018-1521-0) contains supplementary material, which is available to authorized users.

BGPI : équipe 4; International audience; 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.

UMR BFP - Equipe GFDF; International audience; Improved plant varieties are important in our attempts to face the challenges of a growing human population and limited planet resources. Plant breeding relies on meiotic crossovers to combine favourable alleles into elite varieties1. However, meiotic crossovers are relatively rare, typically one to three per chromosome2, limiting the efficiency of the breeding process and related activities such as genetic mapping. Several genes that limit meiotic recombination were identified in the model species Arabidopsis thaliana2. Mutation of these genes in Arabidopsis induces a large increase in crossover frequency. However, it remained to be demonstrated whether crossovers could also be increased in crop species hybrids. We explored the effects of mutating the orthologues of FANCM3, RECQ44 or FIGL15 on recombination in three distant crop species, rice (Oryza sativa), pea (Pisum sativum) and tomato (Solanum lycopersicum). We found that the single recq4 mutation increases crossovers about three-fold in these crops, suggesting that manipulating RECQ4 may be a universal tool for increasing recombination in plants. Enhanced recombination could be used with other state-of-the-art technologies such as genomic selection, genome editing or speed breeding6 to enhance the pace and efficiency of plant improvement.

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., Plant root architecture can adapt to different nutrient conditions in the soil. Here Giri et al. show that the rice auxin influx carrier AUX1 mobilizes auxin from the root apex to the differentiation zone and promotes root hair elongation when roots encounter low external phosphate.

The root system anchors the plant to the soil and contributes to plant autotrophy by taking up nutrients and water. In relation with this nutritional function, root development is largely impacted by availability of nutrients and water. Due to human activity, plants, in particular crops, can also be exposed to pollutants which can be absorbed and incorporated into the food chain. Cesium in soils is present at non-toxic concentrations for the plant (micromolar or less), even in soils highly polluted with radioactive cesium due to nuclear accidents. Here, we report on the morphological response of rice roots to Cs+ at micromolar concentrations. It is shown that Cs+ reduces root elongation without affecting root dry weight. Noteworthy, inactivation of the Cs+-permeable K+ transporter OsHAK1 prevents such effect of Cs+, suggesting that internal Cs+ triggers the modification of the root system.

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.

International audience; The root system anchors the plant to the soil and contributes to plant autotrophy by taking up nutrients and water. In relation with this nutritional function, root development is largely impacted by availability of nutrients and water. Due to human activity, plants, in particular crops, can also be exposed to pollutants which can be absorbed and incorporated into the food chain. Cesium in soils is present at non-toxic concentrations for the plant (micromolar or less), even in soils highly polluted with radioactive cesium due to nuclear accidents. Here, we report on the morphological response of rice roots to Cs+ at micromolar concentrations. It is shown that Cs+ reduces root elongation without affecting root dry weight. Noteworthy, inactivation of the Cs+-permeable K+ transporter OsHAK1 prevents such effect of Cs+, suggesting that internal Cs+ triggers the modification of the root system.

We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis, AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H2O2, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na+ but higher K+ and Ca2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive (NHX1, CAT1, APX1, and DREB1A) or negative (HKT1 and KT1) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.

We evaluated the yields of Oryza sativa L. ‘Nipponbare’ rice lines expressing a gene encoding an A20/AN1 domain stress-associated protein, AlSAP, from the halophyte grass Aeluropus littoralis under the control of different promoters. Three independent field trials were conducted, with drought imposed at the reproductive stage. In all trials, the two transgenic lines, RN5 and RN6, consistently out-performed non-transgenic (NT) and wild-type (WT) controls, providing 50–90% increases in grain yield (GY). Enhancement of tillering and panicle fertility contributed to this improved GY under drought. In contrast with physiological records collected during previous greenhouse dry-down experiments, where drought was imposed at the early tillering stage, we did not observe significant differences in photosynthetic parameters, leaf water potential, or accumulation of antioxidants in flag leaves of AlSAP-lines subjected to drought at flowering. However, AlSAP expression alleviated leaf rolling and leaf drying induced by drought, resulting in increased accumulation of green biomass. Therefore, the observed enhanced performance of the AlSAP-lines subjected to drought at the reproductive stage can be tentatively ascribed to a primed status of the transgenic plants, resulting from a higher accumulation of biomass during vegetative growth, allowing reserve remobilization and maintenance of productive tillering and grain filling. Under irrigated conditions, the overall performance of AlSAP-lines was comparable with, or even significantly better than, the NT and WT controls. Thus, AlSAP expression inflicted no penalty on rice yields under optimal growth conditions. Our results support the use of AlSAP transgenics to reduce rice GY losses under drought conditions.

International audience; As a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA-binding proteins called transcription activator-like effectors (TALEs) that bind to effector-binding elements (EBEs) in a sequence-specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice-Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE-nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild-type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14-inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE-mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.

In the arbuscular mycorrhizal (AM) symbiosis, plants satisfy part of their nitrogen (N) requirement through the AM pathway. In sorghum, the ammonium transporters (AMT) AMT3;1, and to a lesser extent AMT4, are induced in cells containing developing arbuscules. Here, we have characterized orthologs of AMT3;1 and AMT4 in four other grasses in addition to sorghum. AMT3;1 and AMT4 orthologous genes are induced in AM roots, suggesting that in the common ancestor of these five plant species, both AMT3;1 and AMT4 were already present and upregulated upon AM colonization. An artificial microRNA approach was successfully used to downregulate either AMT3;1 or AMT4 in rice. Mycorrhizal root colonization and hyphal length density of knockdown plants were not affected at that time, indicating that the manipulation did not modify the establishment of the AM symbiosis and the interaction between both partners. However, expression of the fungal phosphate transporter FmPT was significantly reduced in knockdown plants, indicating a reduction of the nutrient fluxes from the AM fungus to the plant. The AMT3;1 knockdown plants (but not the AMT4 knockdown plants) were significantly less stimulated in growth by AM fungal colonization, and uptake of both 15N and 33P from the AM fungal network was reduced. This confirms that N and phosphorus nutrition through the mycorrhizal pathway are closely linked. But most importantly, it indicates that AMT3;1 is the prime plant transporter involved in the mycorrhizal ammonium transfer and that its function during uptake of N cannot be performed by AMT4.

The roles of potassium channels from the Shaker family in stomatal movements have been investigated by reverse genetics analyses in Arabidopsis (Arabidopsis thaliana), but corresponding information is lacking outside this model species. Rice (Oryza sativa) and other cereals possess stomata that are more complex than those of Arabidopsis. We examined the role of the outward Shaker K+ channel gene OsK5.2. Expression of the OsK5.2 gene (GUS reporter strategy) was observed in the whole stomatal complex (guard cells and subsidiary cells), root vasculature, and root cortex. In stomata, loss of OsK5.2 functional expression resulted in lack of time-dependent outward potassium currents in guard cells, higher rates of water loss through transpiration, and severe slowdown of stomatal closure. In line with the expression of OsK5.2 in the plant vasculature, mutant plants displayed a reduced K+ translocation from the root system toward the leaves via the xylem. The comparison between rice and Arabidopsis show that despite the strong conservation of Shaker family in plants, substantial differences can exist between the physiological roles of seemingly orthologous genes, as xylem loading depends on SKOR and stomatal closure on GORK in Arabidopsis, whereas both functions are executed by the single OsK5.2 Shaker in rice. Since a waxy cuticle covers outer leaf tissues, water vapor diffusion into the atmosphere occurs mainly through the stomatal pores at the leaf surface. The size of the stomatal aperture is tightly regulated to optimize gas exchanges between the leaf inner tissues and the atmosphere, including CO2 intake for photosynthesis and water loss by transpiration (Lawson and Blatt, 2014). This is achieved by fine tuning of the turgor pressure of the two guard cells that surround the stomatal pore and involves a complex coordinated activity of transport systems at the guard cell plasma membrane and vacuolar membrane (Hedrich, 2012; Chen et al., 2012; Hills et al., 2012; Kollist et al., 2014). This control also affects long-distance transport of mineral nutrients from the roots, which take up these nutrients, to the aerial parts, to support plant growth (Marschner et al., 1996). Potassium ion (K+), as a major inorganic constituent of the plant cells and the most abundant cation in the cytosol, is an essential macronutrient for growth and development. It is involved in various functions, including electrical neutralization of negative charges, control of cell membrane polarization, and osmoregulation (Clarkson and Hanson, 1980; Leigh and Wyn Jones, 1984). K+ is thus the main cation absorbed by the roots and circulating within the plant at the cellular or long-distance levels. In guard cells, it is well known as a major contributor, with Cl-, NO3− and malate, to the osmolarity (Raschke and Schnabl, 1978; Willmer and Fricker, 1996). Stomatal opening is initiated by activation of plasma membrane proton pumps in guard cells, which promotes K+ influx through voltage-gated inward K+ channels, as well as anion uptake through H+-anion symporters (Blatt, 1987a; Schroeder et al., 1987; Roelfsema and Prins, 1997; Talbott and Zeiger, 1998; Guo et al., 2003; Jezek and Blatt, 2017). Conversely, stomatal closure requires inhibition of proton pumping at the guard cell membrane and activation of both anion channels and voltage-gated outward K+ channels. The molecular mechanisms responsible for inward and outward K+ fluxes across the plasma membrane have been extensively investigated in Arabidopsis (Arabidopsis thaliana). Shaker channel subunits, present as a nine-member family in Arabidopsis, have been shown to form the major pathways for these fluxes throughout the plant (Véry and Sentenac, 2003). In the Arabidopsis model species, four genes encoding Shaker channel subunits have been identified as playing a major role in root to shoot K+ translocation and in stomatal movements. The SKOR subunit, which is expressed in root pericycle and xylem parenchyma, forms outwardly rectifying channels involved in K+ secretion into the xylem sap (Gaymard et al., 1998). In stomata, the inward Shaker channel subunits KAT1 and KAT2 are involved in guard cell K+ uptake, and the outward Shaker channel GORK mediates guard cell K+ release (Ache et al., 2000; Pilot et al., 2001; Szyroki et al., 2001; Hosy et al., 2003; Lebaudy et al., 2008). Whereas these Shaker subunits have been deeply characterized in Arabidopsis, and the Shaker family, as a whole, can be considered as the best characterized family of plant membrane transport systems, little information at the molecular genetic level is yet available on this family outside Arabidopsis. The stomatal complex in rice (Oryza sativa), the current model cereal, is very different from that of Arabidopsis (Itoh et al., 2005; Franks and Farquhar, 2007; Roelfsema and Hedrich, 2009). In rice, like in other cereals, it comprises two subsidiary cells in addition to the two guard cells. Substantial differences may thus exist between rice and Arabidopsis in stomatal functioning (Mumm et al., 2011). Rice possesses two putative outward Shaker channel subunits (Pilot et al., 2003; Véry et al., 2014), named OsK5.1 (or OsSKOR) and OsK5.2 (or OsGORK; Pilot et al., 2003; Kim et al., 2015). OsK5.1 was reported to be mainly expressed in root vasculature, just as SKOR in Arabidopsis (Kim et al., 2015). In contrast, OsK5.2 was found to be expressed both in roots and shoots (Kim et al., 2015). The expression pattern of this gene at the tissue level has, however, not been described. Here, we investigate the expression pattern and role of this rice outward Shaker gene. OsK5.2 is shown to play two important roles in rice plants: it mediates K+ translocation into the xylem sap toward the shoots, and it is involved in K+ release from guard cells and stomatal movements.

Summary Occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below μM), cesium (Cs+) can be taken up by crops and transported to their edible parts. Such a plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species, such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side effects. On the other hand, development of biotechnological approaches aiming at reducing Cs+ accumulation in rice remained challenging. Here, we show that inactivation of the Cs+-permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells expressing OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+, and (ii) a high capacity to transport Cs+ from very low external concentrations, which is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137Cs+, plants lacking OsHAK1 function displayed strikingly reduced levels of 137Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents. This article is protected by copyright. All rights reserved.

AGAP : équipe ID / GE2pop; Corrigendum: New insights on leucine-rich repeats receptor-like kinase orthologous relationships in angiosperms

Grain quality is an important agricultural trait that is mainly determined by grain size and composition. Here, we characterize the role of the rice transcription factor (TF) SALT-RESPONSIVE ERF1 (SERF1) during grain development. Through genome-wide expression profiling and chromatin immunoprecipitation, we found that SERF1 directly regulates RICE PROLAMIN-BOX BINDING FACTOR (RPBF), a TF that functions as a positive regulator of grain filling. Loss of SERF1 enhances RPBF expression resulting in larger grains with increased starch content, while SERF1 overexpression represses RPBF resulting in smaller grains. Consistently, during grain filling, starch biosynthesis genes such as GRANULE-BOUND STARCH SYNTHASEI (GBSSI), STARCH SYNTHASEI (SSI), SSIIIa, and ADP-GLUCOSE PYROPHOSPHORYLASE LARGE SUBUNIT2 (AGPL2) are up-regulated in SERF1 knockout grains. Moreover, SERF1 is a direct upstream regulator of GBSSI. In addition, SERF1 negatively regulates germination by controlling RPBF expression, which mediates the gibberellic acid (GA)-induced expression of RICE AMYLASE1A (RAmy1A). Loss of SERF1 results in more rapid seedling establishment, while SERF1 overexpression has the opposite effect. Our study reveals that SERF1 represents a negative regulator of grain filling and seedling establishment by timing the expression of RPBF.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below μm), cesium (Cs

Plant expression of the entomopathogenic bacteria Bacillus thuringiensis cry gene has reduced the damage created by insect pests in several economically important cultures. For this study, we have conducted genetic transformation of the indica rice ‘IRGA 424,’ via Agrobacterium tumefaciens, using the B. thuringiensis cry1Aa and cry1B genes, with the objective of obtaining rice plants resistant to the insect pests from this culture. The gene constructions harbor the promoters maize proteinase inhibitor and ubiquitin. The results showed that high concentration of the hormone 2,4-dichlorophenoxyacetic acid and agarose as the gelling agent helped the production of embryogenic calli for the analyzed cultivar. More than 80% of the obtained transformed plants revealed the integration, using polymerase chain reaction, of the cry1Aa and cry1B genes. Analysis of the expression of the heterologous protein by Western blotting revealed the expression of the Cry1B δelta-endotoxin in IRGA 424 plants transformed...

Depuis l’obtention de la premiere plante transgenique de riz en 1988, de larges progres ont ete accomplis pour manipuler les caracteres d’interet agronomique par ingenierie genetique chez cette plante d’importance alimentaire, economique et culturelle majeure. Les caracteres concernes vont de la resistance aux insectes ravageurs et aux pathogenes fongiques, bacteriens et viraux a la tolerance vis-a-vis des contraintes abiotiques (toxicites et carences minerales, temperature excessive ou basse, exces ou deficit en eau) en passant par les qualites nutritives ou industrielles du grain. Des portions entieres de voies metaboliques peuvent a present etre finement manipulees, permettant par exemple la production de β-carotene (provitamine A) dans l’albumen du grain, qui en est naturellement depourvu, ou l’accumulation des enzymes de la photosynthese C4 dans des tissus foliaires precis. La production de molecules d’interet therapeutique ou industriel dans le grain a aussi ete realisee. Bien que certaines de ces innovations soient pretes a etre transferees dans le champ du riziculteur depuis plusieurs annees, les politiques publiques sont encore hesitantes a franchir le pas. Ce retard relatif a ete mis a profit pour conduire de nombreuses etudes sur les impacts alimentaires et environnementaux lies a la consommation et au deploiement de ces nouvelles cultures.

Rice possesses a fibrous root system made up mainly of adventitious roots also called crown or nodal roots. Roots emerge from the nodes first at the stem base and then from nodes of shoot ramifications. The root system is not easily accessible and therefore difficult to study. For this reason phenotypic breeding for rice varieties with improved root systems remains a challenge. Hence, identification of genetic determinants of root development should considerably improve breeding efficiency. Numerous studies are underway on identifying and characterizing genes controlling root traits on rice and other cereals. In this review, we summarize the latest strategies and some recent results concerning the identification and characterization of root genes and quantitative trait loci affecting root development in rice.

Le riz ( Oryza sativa L.) a le double statut de cereale la plus consommee par l’homme et d’espece modele pour les monocotyledones. Elucider la fonction des genes sous-tendant les principaux caracteres d’interet agronomique chez cette plante contribuerait donc a accelerer non seulement l’amelioration varietale du riz mais aussi celle des autres cereales. La transformation genetique est un outil permettant d’accelerer la decouverte de la fonction des genes et l’innovation varietale. La premiere plante transgenique de riz a ete obtenue des 1988, et, depuis, les technologies ont regulierement evolue vers plus d’efficacite et de precision. Nous presentons ici les grandes etapes de l’evolution de ces techniques, ainsi que leurs applications pour l’analyse fonctionnelle des genes. En particulier, les perspectives ouvertes par de nouveaux sauts technologiques en cours, qui permettent a present une modification ciblee des genomes sans introduction d’acide desoxyribonucleique (ADN) superflu, seront illustrees et discutees.