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.

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 stressresponsive 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.

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.

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialized form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the world’s most productive crops. The C4 cycle is accomplished through cell-type-specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we tested whether mechanisms impacting on transcription in C4 plants evolved from ancestral components found in C3 species. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 Panicoid grass species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C 4 photosynthesis and depends on the cell-specific accumulation of major C 4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyllspecific gene expression in maize, has been shown to keep the same properties when introduced into rice (C 3 plant), indicating that rice has the transcription factors (TFs) needed to confer C 4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C 4 regulators were co-opted from C 3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo-and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells.ZmbHLH80 and ZmbHLH90 antagonistically regulate ZmPEPC1 275 Transactivation assays in rice protoplastsFor the transient expression assay in pL2V-pZMUbi-HYG_pZm-PEPC321-GUS-17244.b rice protoplasts, we used the p2GW7:: OsbHLH112 effector plasmid. To construct this plasmid, the

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Identification of differences between genetically modified plants and their original counterparts plays a central role in risk assessment strategy. Our main goal was to better understand the relevance of transgene presence, genetic, and epigenetic changes induced by transgene insertion, and in vitro culture in putative unintended differences between a transgenic and its comparator. Thus, we have used multiplex fluorescence 2DE coupled with MS to characterize the proteome of three different rice lines (Oryza sativa L. ssp. japonica cv. Nipponbare): a control conventional line (C), an Agrobacterium-transformed transgenic line (Ta) and a negative segregant (NSb). We observed that Ta and NSb appeared identical (with only one spot differentially abundant--fold difference ≥ 1.5), contrasting with the control (49 spots with fold difference ≥ 1.5, in both Ta and NSb vs. control). Given that in vitro culture was the only event in common between Ta and NSb, we hypothesize that in vitro culture stress was the most relevant condition contributing for the observed proteomic differences. MS protein identification support our hypothesis, indicating that Ta and NSb lines adjusted their metabolic pathways and altered the abundance of several stress related proteins in order to cope with in vitro culture.

Cadmium (Cd) was shown to co-localise with calcium (Ca) in oxalate crystals in the stems and leaves of Cd tolerant Gomphrena claussenii, but Cd binding remained unresolved. Using synchrotron radiation X-ray absorption near edge spectroscopy we demonstrate that in oxalate crystals of hydroponically grown G. claussenii the vast majority of Cd is bound to oxygen ligands in oxalate crystals (>88%; Cd-O-C coordination) and the remaining Cd is bound to sulphur ligands (Cd-S-C coordination). Cadmium binding to oxalate does not depend on the amount of Ca supplied or from which organs the crystals originate (stems and mature leaves). By contrast, roots contain no oxalate crystals and therein Cd is bound predominantly by S ligands. The potential to remove Cd by extraction of Cd-rich oxalate crystals from plant material should be tested in phytoextraction or phytomining strategies.