Rice F-bZIP transcription factors regulate the zinc deficiency response.

The F-bZIP transcription factors bZIP19 and bZIP23 are the central regulators of the zinc deficiency response in Arabidopsis, and phylogenetic analysis of F-bZIP homologs across land plants indicates that the regulatory mechanism of the zinc deficiency response may be conserved. Here, we identified the rice F-bZIP homologs and investigated their function. OsbZIP48 and OsbZIP50, but not OsbZIP49, complement the zinc deficiency-hypersensitive Arabidopsis bzip19bzip23 double mutant. Ectopic expression of OsbZIP50 in Arabidopsis significantly increases plant zinc accumulation under control zinc supply, suggesting an altered Zn sensing in OsbZIP50. In addition, we performed a phylogenetic analysis of F-bZIP homologs from representative monocot species that supports the branching of plant F-bZIPs into Group 1 and Group 2. Our results suggest that regulation of the zinc deficiency response in rice is conserved, with OsbZIP48 being a functional homolog of AtbZIP19 and AtbZIP23. A better understanding of the mechanisms behind the Zn deficiency response in rice and other important crops will contribute to develop plant-based strategies to address the problems of Zn deficiency in soils, crops, and cereal-based human diets.

Insights into the transcriptional and post-transcriptional regulation of the rice SUMOylation machinery and into the role of two rice SUMO proteases.

BACKGROUND: SUMOylation is an essential eukaryotic post-translation modification that, in plants, regulates numerous cellular processes, ranging from seed development to stress response. Using rice as a model crop plant, we searched for potential regulatory points that may influence the activity of the rice SUMOylation machinery genes. RESULTS: We analyzed the presence of putative cis-acting regulatory elements (CREs) within the promoter regions of the rice SUMOylation machinery genes and found CREs related to different cellular processes, including hormone signaling. We confirmed that the transcript levels of genes involved in target-SUMOylation, containing ABA- and GA-related CREs, are responsive to treatments with these hormones. Transcriptional analysis in Nipponbare (spp. japonica) and LC-93-4 (spp. indica), showed that the transcript levels of all studied genes are maintained in the two subspecies, under normal growth. OsSUMO3 is an exceptional case since it is expressed at low levels or is not detectable at all in LC-93-4 roots and shoots, respectively. We revealed post-transcriptional regulation by alternative splicing (AS) for all genes studied, except for SUMO coding genes, OsSIZ2, OsOTS3, and OsELS2. Some AS forms have the potential to alter protein domains and catalytic centers. We also performed the molecular and phenotypic characterization of T-DNA insertion lines of some of the genes under study. Knockouts of OsFUG1 and OsELS1 showed increased SUMOylation levels and non-overlapping phenotypes. The fug1 line showed a dwarf phenotype, and significant defects in fertility, seed weight, and panicle architecture, while the els1 line showed early flowering and decreased plant height. We suggest that OsELS1 is an ortholog of AtEsd4, which was also supported by our phylogenetic analysis. CONCLUSIONS: Overall, we provide a comprehensive analysis of the rice SUMOylation machinery and discuss possible effects of the regulation of these genes at the transcriptional and post-transcriptional level. We also contribute to the characterization of two rice SUMO proteases, OsELS1 and OsFUG1.

Regulation of Na+ and K+ homeostasis in plants: towards improved salt stress tolerance in crop plants.

Soil salinity is a major abiotic stress that results in considerable crop yield losses worldwide. However, some plant genotypes show a high tolerance to soil salinity, as they manage to maintain a high K+/Na+ ratio in the cytosol, in contrast to salt stress susceptible genotypes. Although, different plant genotypes show different salt tolerance mechanisms, they all rely on the regulation and function of K+ and Na+ transporters and H+ pumps, which generate the driving force for K+ and Na+ transport. In this review we will introduce salt stress responses in plants and summarize the current knowledge about the most important ion transporters that facilitate intra- and intercellular K+ and Na+ homeostasis in these organisms. We will describe and discuss the regulation and function of the H+-ATPases, H+-PPases, SOS1, HKTs, and NHXs, including the specific tissues where they work and their response to salt stress.

Five novel transcription factors as potential regulators of OsNHX1 gene expression in a salt tolerant rice genotype.

KEY MESSAGE: This manuscript reports the identification and characterization of five transcription factors binding to the promoter of OsNHX1 in a salt stress tolerant rice genotype (Hasawi). Although NHX1 encoding genes are known to be highly regulated at the transcription level by different abiotic stresses, namely salt and drought stress, until now only one transcription factor (TF) binding to its promoter has been reported. In order to unveil the TFs regulating NHX1 gene expression, which is known to be highly induced under salt stress, we have used a Y1H system to screen a salt induced rice cDNA expression library from Hasawi. This approach allowed us to identify five TFs belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) binding to the OsNHX1 gene promoter. We have also shown that these TFs act either as transcriptional activators (OsPCF2, OsNIN-like4) or repressors (OsCPP5, OsNIN-like2) and their encoding genes are differentially regulated by salt and PEG-induced drought stress in two rice genotypes, Nipponbare (salt-sensitive) and Hasawi (salt-tolerant). The transactivation activity of OsNIN-like3 was not possible to determine. Increased soil salinity has a direct impact on the reduction of plant growth and crop yield and it is therefore fundamental to understand the molecular mechanisms underlying gene expression regulation under adverse environmental conditions. OsNHX1 is the most abundant K+-Na+/H+ antiporter localized in the tonoplast and its gene expression is induced by salt, drought and ABA. To investigate how OsNHX1 is transcriptionally regulated in response to salt stress in a salt-tolerant rice genotype (Hasawi), a salt-stress-induced cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsNHX1 promoter as bait. Five transcription factors (TFs) belonging to three distinct TF families: one TCP (OsPCF2), one CPP (OsCPP5) and three NIN-like (OsNIN-like2, OsNIN-like3 and OsNIN-like4) were identified as binding to OsNHX1 promoter. Transactivation activity assays performed in Arabidopsis and rice protoplasts showed that OsPCF2 and OsNIN-like4 are activators of the OsNHX1 gene expression, while OsCPP5 and OsNIN-like2 act as repressors. The transactivation activity of OsNIN-like3 needs to be further investigated. Gene expression studies showed that OsNHX1 transcript level is highly induced by salt and PEG-induced drought stress in both shoots and roots in both Nipponbare and Hasawi rice genotypes. Nevertheless, OsNHX1 seems to play a particular role in shoots in response to drought. Most of the TFs binding to OsNHX1 promoter showed a modest transcriptional regulation under stress conditions, however, in response to most of the conditions studied, the OsPCF2 was induced earlier than OsNHX1, indicating that OsPCF2 may activate OsNHX1 gene expression. In addition, although the OsNHX1 response to salt and PEG-induced drought stress in either shoots or roots was quite similar in both rice genotypes, the expression of OsPCF2 in roots under salt stress and the OsNIN-like4 in roots subjected to PEG was mainly up-regulated in Hasawi, indicating that these TFs may be associated with the salt and drought stress tolerance observed for this genotype.

Regulation of Na+ and K+ homeostasis in plants: towards improved salt stress tolerance in crop plants

Abstract Soil salinity is a major abiotic stress that results in considerable crop yield losses worldwide. However, some plant genotypes show a high tolerance to soil salinity, as they manage to maintain a high K+/Na+ ratio in the cytosol, in contrast to salt stress susceptible genotypes. Although, different plant genotypes show different salt tolerance mechanisms, they all rely on the regulation and function of K+ and Na+ transporters and H+ pumps, which generate the driving force for K+ and Na+ transport. In this review we will introduce salt stress responses in plants and summarize the current knowledge about the most important ion transporters that facilitate intra- and intercellular K+ and Na+ homeostasis in these organisms. We will describe and discuss the regulation and function of the H+-ATPases, H+-PPases, SOS1, HKTs, and NHXs, including the specific tissues where they work and their response to salt stress.

Screening for Abiotic Stress Tolerance in Rice: Salt, Cold, and Drought.

Rice (Oryza sativa) is the primary source of food for more than half of the world population. Most rice varieties are severely injured by abiotic stresses, with strong social and economic impact. Understanding rice responses to stress may help breeding for more tolerant varieties. However, papers dealing with stress experiments often describe very different experimental designs, thus making comparisons difficult. The use of identical setups is the only way to generate comparable data. This chapter is organized into three sections, describing the experimental conditions established at the Genomics of Plant Stress (GPlantS) unit of ITQB to assess the response of rice plants to three different abiotic stresses--high salinity, cold stress, and drought. All sections include a detailed description of the materials and methodology, as well as useful notes gathered from the GPlantS team's experience. We use rice seedlings as plants at this stage show high sensitivity to abiotic stresses. For the salt and cold stress assays we use hydroponic cultures, while for the drought assay plants are grown in soil and subjected to water withholding. All setups enable visual score determination and are suitable for sample collection along the imposition of stress. The proposed methodologies are simple and affordable to implement in most labs, allowing the discrimination of several rice genotypes at the molecular and phenotypic level.

OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors.

High salinity causes remarkable losses in rice productivity worldwide mainly because it inhibits growth and reduces grain yield. To cope with environmental changes, plants evolved several adaptive mechanisms, which involve the regulation of many stress-responsive genes. Among these, we have chosen OsRMC to study its transcriptional regulation in rice seedlings subjected to high salinity. Its transcription was highly induced by salt treatment and showed a stress-dose-dependent pattern. OsRMC encodes a receptor-like kinase described as a negative regulator of salt stress responses in rice. To investigate how OsRMC is regulated in response to high salinity, a salt-induced rice cDNA expression library was constructed and subsequently screened using the yeast one-hybrid system and the OsRMC promoter as bait. Thereby, two transcription factors (TFs), OsEREBP1 and OsEREBP2, belonging to the AP2/ERF family were identified. Both TFs were shown to bind to the same GCC-like DNA motif in OsRMC promoter and to negatively regulate its gene expression. The identified TFs were characterized regarding their gene expression under different abiotic stress conditions. This study revealed that OsEREBP1 transcript level is not significantly affected by salt, ABA or severe cold (5 °C) and is only slightly regulated by drought and moderate cold. On the other hand, the OsEREBP2 transcript level increased after cold, ABA, drought and high salinity treatments, indicating that OsEREBP2 may play a central role mediating the response to different abiotic stresses. Gene expression analysis in rice varieties with contrasting salt tolerance further suggests that OsEREBP2 is involved in salt stress response in rice.