Tiago Lourenço

Transcription factors and regulation of photosynthetic and related metabolism under environmental stresses.

BACKGROUND Environmental conditions, such as water supply, temperature and salinity, strongly affect plant growth and development. Extremes of these conditions (abiotic stresses) adversely affect many different mechanisms associated with plant responses and adaptation to stress: photosynthetic mechanisms, e.g. stomatal control of CO(2) diffusion, photosystem II repair, ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity and scavenging of reactive oxygen species (ROS), are susceptible to damage, and photosynthetic efficiency can be greatly decreased. Responses and adaptations require differential gene expression, which is regulated by specific transcription factors (TFs). SCOPE The role and regulation of several TFs involved in abiotic stress response pathways are considered, with emphasis on new findings regarding expression of genes related to both stomatal and non-stomatal limitations to CO(2) photosynthetic assimilation. CONCLUSIONS Many TFs, belonging to different families (e.g. MYB, bZIP and DREB), have been related to abiotic stress responses; however, only a few are known to regulate the expression of photosynthesis-related genes in response to stress. Several TFs belonging to the MYB family play an important role in both stomatal and non-stomatal responses by regulation of stomatal numbers and sizes, and metabolic components, respectively. To obtain more insight into this area of potentially large agronomic impact, it is essential to identify and functionally characterize new TFs that mediate the stress responses regulating the expression of genes associated with photosynthesis and related metabolism.

Microarray analyses reveal that plant mutagenesis may induce more transcriptomic changes than transgene insertion

Controversy regarding genetically modified (GM) plants and their potential impact on human health contrasts with the tacit acceptance of other plants that were also modified, but not considered as GM products (e.g., varieties raised through conventional breeding such as mutagenesis). What is beyond the phenotype of these improved plants? Should mutagenized plants be treated differently from transgenics? We have evaluated the extent of transcriptome modification occurring during rice improvement through transgenesis versus mutation breeding. We used oligonucleotide microarrays to analyze gene expression in four different pools of four types of rice plants and respective controls: (i) a γ-irradiated stable mutant, (ii) the M1 generation of a 100-Gy γ-irradiated plant, (iii) a stable transgenic plant obtained for production of an anticancer antibody, and (iv) the T1 generation of a transgenic plant produced aiming for abiotic stress improvement, and all of the unmodified original genotypes as controls. We found that the improvement of a plant variety through the acquisition of a new desired trait, using either mutagenesis or transgenesis, may cause stress and thus lead to an altered expression of untargeted genes. In all of the cases studied, the observed alteration was more extensive in mutagenized than in transgenic plants. We propose that the safety assessment of improved plant varieties should be carried out on a case-by-case basis and not simply restricted to foods obtained through genetic engineering.

Transcription Regulation of Abiotic Stress Responses in Rice: A Combined Action of Transcription Factors and Epigenetic Mechanisms

Plant growth and crop production are highly reduced by adverse environmental conditions and rice is particularly sensitive to abiotic stresses. Plants have developed a number of different mechanisms to respond and try to adapt to abiotic stress. Plant response to stress such as drought, cold, and high salinity, implies rapid and coordinated changes at transcriptional level of entire gene networks. During the last decade many transcription factors, belonging to different families, have been shown to act as positive or negative regulators of stress responsive genes, thus playing an extremely important role in stress signaling. More recently, epigenetic mechanisms have been also involved in the regulation of the stress responsive genes. In this review, we have performed a comprehensive analysis of the rice transcription factors reported so far as being involved in abiotic stress responses. The impact of abiotic stresses on epigenomes is also addressed. Finally, we update the connections made so far between DNA-binding transcription factors (TFs), and epigenetic mechanisms (DNA methylation and histones methylation or acetylation) emphasizing an integrative view of transcription regulation.

Seven zinc-finger transcription factors are novel regulators of the stress responsive gene OsDREB1B

Plants have evolved several mechanisms in order to cope with adverse environmental conditions. The transcription factors (TFs) belonging to the DREB1/CBF subfamily have been described as major regulators of the plant responses to different abiotic stresses. This study focused on the rice gene OsDREB1B, initially described as highly and specifically induced by cold. However, here it is shown that OsDREB1B is not only induced by low temperatures, but also by drought and mechanical stress. In order to identify novel TFs that bind to its promoter, a yeast one-hybrid system was used to screen a cold-induced cDNA expression library. Thereby seven novel Zn-finger TFs were identified that bind to the promoter of OsDREB1B. Among them, there were four Zn-finger homeodomain (ZF-HD) and three C(2)H(2)-type Zn-finger TFs. Gene expression studies showed that these TFs are differentially regulated at transcriptional level by different abiotic stress conditions, which is illustrative of the crosstalk between stress signalling pathways. Protein-protein interaction studies revealed the formation of homo- and heterodimers among the ZF-HD TFs identified, but not for the C(2)H(2)-type. Using a transactivation assay in Arabidopsis protoplasts, all the TFs identified repressed the expression of the reporter gene, driven by the promoter of OsDREB1B. This assay also showed that the dimerization observed between the ZF-HD TFs may play a role on their transactivation activity. The results here presented suggest a prominent role of Zn-finger TFs in the regulation of OsDREB1B.

Isolation and characterization of rice (Oryza sativa L.) E3-ubiquitin ligase OsHOS1 gene in the modulation of cold stress response.

Plants can cope with adverse environmental conditions through the activation of stress response signalling pathways, in which the proteasome seems to play an important role. However, the mechanisms underlying the proteasome-mediated stress response in rice are still not fully understood. To address this issue, we have identified a rice E3-ubiquitin ligase, OsHOS1, and characterized its role in the modulation of the cold stress response. Using a RNA interference (RNAi) transgenic approach we found that, under cold conditions, the RNAi::OsHOS1 plants showed a higher expression level of OsDREB1A. This was correlated with an increased amount of OsICE1, a master transcription factor of the cold stress signalling. However, the up-regulation of OsDREB1A was transient and the transgenic plants did not show increased cold tolerance. Nevertheless, we could confirm the interaction of OsHOS1 with OsICE1 by Yeast-Two hybrid and bi-molecular fluorescence complementation in Arabidopsis protoplasts. Moreover, we could also determine through an in vitro degradation assay that the higher amount of OsICE1 in the transgenic plants was correlated with a lower amount of OsHOS1. Hence, we could confirm the involvement of the proteasome in this response mechanism. Taken together our results confirm the importance of OsHOS1, and thus of the proteasome, in the modulation of the cold stress signalling in rice.

Rice phytochrome-interacting factor protein OsPIF14 represses OsDREB1B gene expression through an extended N-box and interacts preferentially with the active form of Phytochrome B

DREB1/CBF genes, known as major regulators of plant stress responses, are rapidly and transiently induced by low temperatures. Using a yeast one-hybrid screening, we identified a putative Phytochrome-Interacting bHLH Factor (OsPIF14), as binding to the OsDREB1B promoter. bHLH proteins are able to bind to hexameric E-box (CANNTG) or N-box (CACG(A/C)G) motifs, depending on transcriptional activity. We have shown that OsPIF14 binds to the OsDREB1B promoter through two N-boxes and that the flanking regions of the hexameric core are essential for protein-DNA interaction and stability. We also showed that OsPIF14 down-regulates OsDREB1B gene expression in rice protoplasts, corroborating the OsPIF14 repressor activity observed in the transactivation assays using Arabidopsis protoplasts. In addition, we showed that OsPIF14 is indeed a phytochrome interacting factor, which preferentially binds to the active form (Pfr) of rice phytochrome B. This raises the possibility that OsPIF14 activity might be modulated by light. However, we did not observe any regulation of the OsDREB1B gene expression by light under control conditions. Moreover, OsPIF14 gene expression was shown to be modulated by different treatments, such as drought, salt, cold and ABA. Interestingly, OsPIF14 showed also a specific cold-induced alternative splicing. All together, these results suggest the possibility that OsPIF14 is involved in cross-talk between light and stress signaling through interaction with the OsDREB1B promoter. Although in the absence of stress, OsDREB1B gene expression was not regulated by light, given previous reports, it remains possible that OsPIF14 has a role in light modulation of stress responses.

Inducible and constitutive expression of HvCBF4 in rice leads to differential gene expression and drought tolerance

The effects of the ectopic expression of a barley transcription factor (HvCBF4) under the control of a constitutive (maize Ubi1) or a stress-inducible (Arabidopsis RD29A) promoter in the abiotic stress response in rice (Oryza sativa L.) was investigated. The transformed plants were analyzed both at molecular and physiological level and the AtRD29A::HvCBF4 plants were further analyzed using the GeneChip® rice genome array under control conditions. Only the plants constitutively expressing HvCBF4 have shown increased survival to drought stress, but not to cold or high-salinity. These plants have also shown better photosynthetic capacity, as determined by chlorophyll fluorescence. Plants expressing AtRD29A::HvCBF4 did not show increased survival to any of the stresses applied. However in the GeneChip® microarray, these plants have shown up-regulation of many stress-responsive genes (> 400) as compared to non-transformed plants. Interestingly, RT-PCR analysis revealed not only differential gene expression between roots and shoots, but also between transgenic lines with the different promoters. Our results indicate that different HvCBF4 expression levels resulted in different transcriptomes and drought tolerance. Given that AtRD29A::HvCBF4 plants did not show increased tolerance to any of the imposed stresses, we may conclude that this promoter may be inappropriate for rice transformation aiming for enhanced abiotic stress tolerance.

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 teams 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.

Expression of prune dwarf Ilarvirus coat protein sequences in Nicotiana benthamiana plants interferes with PDV systemic proliferation

Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.

The Rice E3-Ubiquitin Ligase HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 Modulates the Expression of ROOT MEANDER CURLING, a Gene Involved in Root Mechanosensing, through the Interaction with Two ETHYLENE-RESPONSE FACTOR Transcription Factors

Rice root curling, a response to a mechanical barrier that involves the plant hormone jasmonic acid, is modulated by an E3-ubiquitin ligase. Plant roots can sense and respond to a wide diversity of mechanical stimuli, including touch and gravity. However, little is known about the signal transduction pathways involved in mechanical stimuli responses in rice (Oryza sativa). This work shows that rice root responses to mechanical stimuli involve the E3-ubiquitin ligase rice HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENE1 (OsHOS1), which mediates protein degradation through the proteasome complex. The morphological analysis of the roots in transgenic RNA interference::OsHOS1 and wild-type plants, exposed to a mechanical barrier, revealed that the OsHOS1 silencing plants keep a straight root in contrast to wild-type plants that exhibit root curling. Moreover, it was observed that the absence of root curling in response to touch can be reverted by jasmonic acid. The straight root phenotype of the RNA interference::OsHOS1 plants was correlated with a higher expression rice ROOT MEANDER CURLING (OsRMC), which encodes a receptor-like kinase characterized as a negative regulator of rice root curling mediated by jasmonic acid. Using the yeast two-hybrid system and bimolecular fluorescence complementation assays, we showed that OsHOS1 interacts with two ETHYLENE-RESPONSE FACTOR transcription factors, rice ETHYLENE-RESPONSIVE ELEMENT BINDING PROTEIN1 (OsEREBP1) and rice OsEREBP2, known to regulate OsRMC gene expression. In addition, we showed that OsHOS1 affects the stability of both transcription factors in a proteasome-dependent way, suggesting that this E3-ubiquitin ligase targets OsEREBP1 and OsEREBP2 for degradation. Our results highlight the function of the proteasome in rice response to mechanical stimuli and in the integration of these signals, through hormonal regulation, into plant growth and developmental programs.