Delphine Mieulet

Diversity in expression patterns and functional properties in the rice HKT transporter family.

Plant growth under low K+ availability or salt stress requires tight control of K+ and Na+ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K+ Transporters), permeable either to K+ and Na+ or to Na+ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na+ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na+ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na+-K+ symport, Na+ uniport, or inhibited states, depending on external Na+ and K+ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K+ and Na+ accumulation in monocots.

SALT-RESPONSIVE ERF1 Regulates Reactive Oxygen Species–Dependent Signaling during the Initial Response to Salt Stress in Rice

Salinity is a common environmental constraint that is rapidly recognized by plants. This work demonstrates that early sensing of salt stress in rice involves a ROS-mediated response, in which the transcription factor SERF1 and the mitogen-activated protein kinase MAPK5 play a central role. Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified SALT-RESPONSIVE ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H2O2) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance–mediating TFs. Furthermore, we show that SERF1-dependent genes are H2O2 responsive and demonstrate that SERF1 binds to the promoters of MAPK KINASE KINASE6 (MAP3K6), MAPK5, DEHYDRATION-RESPONSIVE ELEMENT BINDING2A (DREB2A), and ZINC FINGER PROTEIN179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species–activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance.

Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library

We characterized the insertion sites of newly transposed copies of the tissue-culture-induced ty1-copia retrotransposon Tos17 in the Oryza Tag Line (OTL) T-DNA mutant library of rice cv. Nipponbare. While Nipponbare contains two native copies of Tos17 the number of additional copies, deduced from Southern blot analyses in a subset of 384 T-DNA lines and using a reverse transcriptase probe specific to the element, ranged from 1 to 8 and averaged 3.37. These copies were shown to be stably inherited and to segregate independently in the progenies of insertion lines. We took advantage of the absence of EcoRV restriction sites in the immediate vicinity of the 3′ LTR of the native copies of Tos17 in the genome sequence of cv. Nipponbare, thereby preventing amplification of corresponding PCR fragments, to efficiently and selectively amplify and sequence flanking regions of newly transposed Tos17 inserts. From 25,286 T-DNA plants, we recovered 19,252 PCR products (76.1%), which were sequenced yielding 14,513 FSTs anchored on the rice pseudomolecules. Following elimination of redundant sequences due to the presence of T-DNA plants deriving from the same cell lineage, these FSTs corresponded to 11,689 unique insertion sites. These unique insertions exhibited higher densities in subtelomeric regions of the chromosomes and hot spots for integration, following a distribution that remarkably paralleled that of Tos17 sites in the National Institute for Agrobiological Sciences (NIAS) library. The insertion sites were mostly found in genic regions (77.5%) and preferably in coding sequences (68.8%) compared to unique T-DNA insertion sites in the same materials (49.1% and 28.3%, respectively). Predicted non- transposable element (TE) genes prone to a high frequency of Tos17 integration (i.e. from 5 to 121 inserts) in the OTL T-DNA collection were generally found to be also hot spots for integration in the NIAS library. The 9,060 Tos17 inserts inserted into non TE genes were found to disrupt a total of 2,773 genes with an average of 3.27 inserts per gene, similar to that in the NIAS library (3.28 inserts per gene on average) whereas the 4,472 T-DNA inserted into genes in the same materials disrupted a total of 3,911 genes (1.14 inserts per gene on average). Interestingly, genes disrupted by both Tos17 and T-DNA inserts in the library represented only 14.9% and 10.6% of the complement of genes interrupted by Tos17 and T-DNA inserts respectively while 52.1% of the genes tagged by Tos17 inserts in the OTL library were found to be tagged also in the NIAS Tos17 library. We concluded that the first advantage in characterizing Tos17 inserts in a rice T-DNA collection lies in a complementary tagging of novel genes and secondarily in finding other alleles in a same genetic background, thereby greatly enhancing the library genome coverage and its overall value for implementing forward and reverse genetics strategies.

Transcription factor OsHsfC1b regulates salt tolerance and development in Oryza sativa ssp. japonica.

The paper describes the functional analysis of a class C heat shock transcription factor from rice (Oryza sativa). OsHsfC1b is shown to play a role in ABA-mediated salt stress tolerance and is required for plant growth under non-stress conditions.

The rice monovalent cation transporter OsHKT2;4: revisited ionic selectivity

The family of plant membrane transporters named HKT (for high-affinity K+ transporters) can be subdivided into subfamilies 1 and 2, which, respectively, comprise Na+-selective transporters and transporters able to function as Na+-K+ symporters, at least when expressed in yeast (Saccharomyces cerevisiae) or Xenopus oocytes. Surprisingly, a subfamily 2 member from rice (Oryza sativa), OsHKT2;4, has been proposed to form cation/K+ channels or transporters permeable to Ca2+ when expressed in Xenopus oocytes. Here, OsHKT2;4 functional properties were reassessed in Xenopus oocytes. A Ca2+ permeability through OsHKT2;4 was not detected, even at very low external K+ concentration, as shown by highly negative OsHKT2;4 zero-current potential in high Ca2+ conditions and lack of sensitivity of OsHKT2;4 zero-current potential and conductance to external Ca2+. The Ca2+ permeability previously attributed to OsHKT2;4 probably resulted from activation of an endogenous oocyte conductance. OsHKT2;4 displayed a high permeability to K+ compared with that to Na+ (permeability sequence: K+ > Rb+ ≈ Cs+ > Na+ ≈ Li+ ≈ NH4+). Examination of OsHKT2;4 current sensitivity to external pH suggested that H+ is not significantly permeant through OsHKT2;4 in most physiological ionic conditions. Further analyses in media containing both Na+ and K+ indicated that OsHKT2;4 functions as K+-selective transporter at low external Na+, but transports also Na+ at high (>10 mm) Na+ concentrations. These data identify OsHKT2;4 as a new functional type in the K+ and Na+-permeable HKT transporter subfamily. Furthermore, the high permeability to K+ in OsHKT2;4 supports the hypothesis that this system is dedicated to K+ transport in the plant.

Expression of the Aeluropus littoralis AlSAP gene in rice confers broad tolerance to abiotic stresses through maintenance of photosynthesis

The expression of AlSAP, in rice cv. Nipponbare, enhances plant tolerance to cold, drought and salt stresses. AlSAP lines showed 100% survival rate and set seeds while control plants did not recover from the cold treatment. Under a severe drought stress treatment (fraction of transpirable soil water down to 0.1), AlSAP lines exhibited enhanced Transpiration Efficiency (TE) and maintained a high A (Assimilation rate) value (22 µmol·m(-2)  s(-1) ) while these values dramatically decreased (A = 4 µmol·m(-2)  s(-1) ) in control plants which were subsequently unable to recover from the stress. Of noteworthy is that AlSAP rice plants yielded a similar and a 60% seed set under control and stress conditions respectively, with regard to wild-type (WT) plants grown under control conditions. This indicates that AlSAP expression imposes no yield penalty and allows seed production even following a severe drought stress at the vegetative stage. Furthermore, AlSAP rice was shown to accumulate transcripts of a pilot set of eight stress-related genes at a significantly higher level than WT plants, both under control and stressed conditions. The results suggest that AlSAP expression generates stress tolerance in plants through maintenance of the photosynthetic apparatus integrity and by stimulating an endogenous adaptive potential which is not effectively accomplished in WT plants.

MULTIPASS, a rice R2R3-type MYB transcription factor, regulates adaptive growth by integrating multiple hormonal pathways

Growth regulation is an important aspect of plant adaptation during environmental perturbations. Here, the role of MULTIPASS (OsMPS), an R2R3-type MYB transcription factor of rice, was explored. OsMPS is induced by salt stress and expressed in vegetative and reproductive tissues. Over-expression of OsMPS reduces growth under non-stress conditions, while knockdown plants display increased biomass. OsMPS expression is induced by abscisic acid and cytokinin, but is repressed by auxin, gibberellin and brassinolide. Growth retardation caused by OsMPS over-expression is partially restored by auxin application. Expression profiling revealed that OsMPS negatively regulates the expression of EXPANSIN (EXP) and cell-wall biosynthesis as well as phytohormone signaling genes. Furthermore, the expression of OsMPS-dependent genes is regulated by auxin, cytokinin and abscisic acid. Moreover, we show that OsMPS is a direct upstream regulator of OsEXPA4, OsEXPA8, OsEXPB2, OsEXPB3, OsEXPB6 and the endoglucanase genes OsGLU5 and OsGLU14. The multiple responses of OsMPS and its target genes to various hormones suggest an integrative function of OsMPS in the cross-talk between phytohormones and the environment to regulate adaptive growth.

Members of BTB Gene Family of Scaffold Proteins Suppress Nitrate Uptake and Nitrogen Use Efficiency

Under N deficiency, in both rice and Arabidopsis, BTs are negative regulators of nitrate uptake and nitrogen utilization efficiency Development of crops with improved nitrogen use efficiency (NUE) is essential for sustainable agriculture. However, achieving this goal has proven difficult since NUE is a complex trait encompassing physiological and developmental processes. We thought to tackle this problem by taking a systems biology approach to identify candidate target genes. First, we used a supervised machine-learning algorithm to predict a NUE gene network in Arabidopsis (Arabidopsis thaliana). Second, we identified BT2, a member of the Bric-a-Brac/Tramtrack/Broad gene family, as the most central and connected gene in the NUE network. Third, we experimentally tested BT2 for a role in NUE. We found NUE decreased in plants overexpressing BT2 gene compared to wild-type plants under limiting nitrate conditions. In addition, NUE increased compared to wild-type plants under low nitrate conditions in double mutant plants in bt2 and its closely related homolog bt1, indicating a functional redundancy of BT1 and BT2 for NUE. Expression of the nitrate transporter genes NRT2.1 and NRT2.4 increased in the bt1/bt2 double mutant compared to wild-type plants, with a concomitant 65% increase in nitrate uptake under low nitrate conditions. Similar to Arabidopsis, we found that mutation of the BT1/BT2 ortholog gene in rice (Oryza sativa) OsBT increased NUE by 20% compared to wild-type rice plants under low nitrogen conditions. These results indicate BT gene family members act as conserved negative regulators of nitrate uptake genes and NUE in plants and highlight them as prime targets for future strategies to improve NUE in crops.

Production of cecropin A antimicrobial peptide in rice seed endosperm

BackgroundCecropin A is a natural antimicrobial peptide that exhibits rapid, potent and long-lasting lytic activity against a broad spectrum of pathogens, thus having great biotechnological potential. Here, we report a system for producing bioactive cecropin A in rice seeds.ResultsTransgenic rice plants expressing a codon-optimized synthetic cecropin A gene drived by an endosperm-specific promoter, either the glutelin B1 or glutelin B4 promoter, were generated. The signal peptide sequence from either the glutelin B1 or the glutelin B4 were N-terminally fused to the coding sequence of the cecropin A. We also studied whether the presence of the KDEL endoplasmic reticulum retention signal at the C-terminal has an effect on cecropin A subcellular localization and accumulation. The transgenic rice plants showed stable transgene integration and inheritance. We show that cecropin A accumulates in protein storage bodies in the rice endosperm, particularly in type II protein bodies, supporting that the glutelin N-terminal signal peptides play a crucial role in directing the cecropin A to this organelle, independently of being tagged with the KDEL endoplasmic reticulum retention signal. The production of cecropin A in transgenic rice seeds did not affect seed viability or seedling growth. Furthermore, transgenic cecropin A seeds exhibited resistance to infection by fungal and bacterial pathogens (Fusarium verticillioides and Dickeya dadantii, respectively) indicating that the in planta-produced cecropin A is biologically active.ConclusionsRice seeds can sustain bioactive cecropin A production and accumulation in protein bodies. The system might benefit the production of this antimicrobial agent for subsequent applications in crop protection and food preservation.

Marker-free transgenic durum wheat cv. Karim expressing the AlSAP gene exhibits a high level of tolerance to salinity and dehydration stresses

We have recently isolated the AlSAP (stress-associated protein) gene from the halophyte grass Aeluropus littoralis and demonstrated that AlSAP expression improves tolerance to continuous salt and drought stresses in transgenic tobacco. To extend these findings to an important crop, we generated marker-free transgenic durum wheat plants of the commercial cv. Karim expressing the AlSAP gene. The integration and expression of AlSAP in T3 homozygous plants were ascertained by Southern, Northern and Western blotting respectively. AlSAP wheat lines exhibited improved germination rates and biomass production under severe salinity and osmotic stress conditions. Following a long-term salt or drought stress greenhouse trial, AlSAP lines produced normally filled grains whereas wild-type (WT) plants either died at the vegetative stage under salt stress or showed markedly reduced grain filling under drought stress. Measurements of the RWC (relative water content) and endogenous Na+ and K+ levels in leaves of AlSAP plants, showed a lower water loss rate and a higher Na+ accumulation in senescent-basal leaves, respectively, compared to those of WT plants. Taken together, these results extend to cereals the high potential of the AlSAP gene for engineering effective drought and salt tolerance.