Diminished Auxin Signaling Triggers Cellular Reprogramming by Inducing a Regeneration Factor in the Liverwort Marchantia polymorpha.

Regeneration in land plants is accompanied by the establishment of new stem cells, which often involves reactivation of the cell division potential in differentiated cells. The phytohormone auxin plays pivotal roles in this process. In bryophytes, regeneration is enhanced by the removal of the apex and repressed by exogenously applied auxin, which has long been proposed as a form of apical dominance. However, the molecular basis behind these observations remains unexplored. Here, we demonstrate that in the liverwort Marchantia polymorpha, the level of endogenous auxin is transiently decreased in the cut surface of decapitated explants, and identify by transcriptome analysis a key transcription factor gene, LOW-AUXIN RESPONSIVE (MpLAXR), which is induced upon auxin reduction. Loss of MpLAXR function resulted in delayed cell cycle reactivation, and transient expression of MpLAXR was sufficient to overcome the inhibition of regeneration by exogenously applied auxin. Furthermore, ectopic expression of MpLAXR caused cell proliferation in normally quiescent tissues. Together, these data indicate that decapitation causes a reduction of auxin level at the cut surface, where, in response, MpLAXR is up-regulated to trigger cellular reprogramming. MpLAXR is an ortholog of Arabidopsis ENHANCER OF SHOOT REGENERATION 1/DORNRÖSCHEN, which has dual functions as a shoot regeneration factor and a regulator of axillary meristem initiation, the latter of which requires a low auxin level. Thus, our findings provide insights into stem cell regulation as well as apical dominance establishment in land plants.

The VFH1 (YLL056C) promoter is vanillin-inducible and enables mRNA translation despite pronounced translation repression caused by severe vanillin stress in Saccharomyces cerevisiae.

Vanillin, furfural and 5-hydroxymethylfurfural (HMF) are representative fermentation inhibitors generated during the pretreatment process of lignocellulosic biomass in bioethanol production. These biomass conversion inhibitors, particularly vanillin, are known to repress translation activity in Saccharomyces cerevisiae. We have reported that the mRNAs of ADH7 and BDH2 were efficiently translated under severe vanillin stress despite marked repression of overall protein synthesis. In this study, we found that expression of VFH1 (YLL056C) was also significantly induced at the protein level by severe vanillin stress. Additionally, we demonstrated that the VFH1 promoter enabled the protein synthesis of other genes including GFP and ALD6 under severe vanillin stress. It is known that transcriptional activation of VFH1 is induced by furfural and HMF, and we verified that Vfh1 protein synthesis was also induced by furfural and HMF. The null mutant of VFH1 delayed growth in the presence of vanillin, furfural and HMF, indicating the importance of Vfh1 for sufficient tolerance against these inhibitors. The protein levels of Vfh1 induced by the inhibitors tested were markedly higher than those of Adh7 and Bdh2, suggesting the superior utility of the VFH1 promoter over the ADH7 or BDH2 promoter for breeding optimized yeast strains for bioethanol production from lignocellulosic biomass.

The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin.

Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccharomyces cerevisiae.

Vanillin and furfural are derived from lignocellulosic biomass and inhibit yeast growth and fermentation as biomass conversion inhibitors. Furfural has been shown to induce oxidative stress in Saccharomyces cerevisiae. Since there has been no report on the relationship between vanillin and oxidative stress, we investigated whether vanillin caused oxidative stress in yeast cells. We showed that vanillin caused the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, and subsequent transcriptional activation of Yap1-target genes. The growth of the null mutant of the YAP1 gene (yap1Δ) was delayed in the presence of vanillin, which indicated that Yap1 plays a role in the acquisition of tolerance to vanillin. We also demonstrated that vanillin facilitated the fragmentation of mitochondria. These findings suggest that the toxicity of vanillin involves damage induced by oxidative stress.

Vanillin inhibits translation and induces messenger ribonucleoprotein (mRNP) granule formation in saccharomyces cerevisiae: application and validation of high-content, image-based profiling.

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

Biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger RNP granules and attenuate translation activity in Saccharomyces cerevisiae.

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

Acidic stress induces the formation of P-bodies, but not stress granules, with mild attenuation of bulk translation in Saccharomyces cerevisiae.

The stress response of eukaryotic cells often causes an attenuation of bulk translation activity and the accumulation of non-translating mRNAs into cytoplasmic mRNP (messenger ribonucleoprotein) granules termed cytoplasmic P-bodies (processing bodies) and SGs (stress granules). We examined effects of acidic stress on the formation of mRNP granules compared with other forms of stress such as glucose deprivation and a high Ca²âº level in Saccharomyces cerevisiae. Treatment with lactic acid clearly caused the formation of P-bodies, but not SGs, and also caused an attenuation of translation initiation, albeit to a lesser extent than glucose depletion. P-body formation was also induced by hydrochloric acid and sulfuric acid. However, lactic acid in SD (synthetic dextrose) medium with a pH greater than 3.0, propionic acid and acetic acid did not induce P-body formation. The results of the present study suggest that the assembly of yeast P-bodies can be induced by external conditions with a low pH and the threshold was around pH 2.5. The P-body formation upon acidic stress required Scd6 (suppressor of clathrin deficiency 6), a component of P-bodies, indicating that P-bodies induced by acidic stress have rules of assembly different from those induced by glucose deprivation or high Ca²âº levels.

Metalloprotease-dependent onset of blood circulation in zebrafish.

The primitive blood circulation requires intravascular plasma flow. However, it remains unclear whether the onset of earliest blood circulation is dependent solely on establishment of a functional circulatory organ or whether it also requires active processes inherent in blood cells. In this study, we present novel mechanisms for the onset of blood circulation by monitoring fluorescently labeled blood precursors and blood vessels in zebrafish. The earliest blood circulation occurs synchronously. This synchrony is achieved by the retention of erythroid precursors on the lumen of the vasculature after their invasion from the subaortic region, and then by simultaneous release of these precursors into the flow. Morphological and biochemical analyses suggest that the onset of blood circulation accompanies disruption of blood cell-to-vessel adhesion and requires metalloprotease-dependent processes. ADAM8, a member of the a disintegrin and metalloprotease (ADAM) family, mediates the onset of blood circulation. In ADAM8-depleted embryos, erythroid cells fail to detach from the vascular lumen and stagnate. Expression of a protease-defective ADAM8 in erythroid cells causes dominant-negative effects on blood circulation, suggesting cell-autonomous roles of ADAM8. Based on these findings, we propose that the first erythroid cells require both flow-dependent passive and proteolysis-dependent active processes to enter the circulation.