Paul Rosas-Santiago

Main conclusion Osmotic stress–induced Ca²⁺ accumulation promotes the formation of specific RFP-ATG8i labeled autophagosome near the hypocotyl-root transition zone, suggesting ATG8i-dependent autophagy or reticulophagy, as a mechanism to alleviate osmotic and endoplasmic reticulum stress in plants. Abstract Autophagy is a crucial and evolutionarily conserved process that breaks down and recycles damaged or unnecessary cytoplasmic components. This process is essential for plant growth and for responding to environmental stresses. In this study, we investigated how autophagy is regulated during osmotic stress in Arabidopsis thaliana, with a focus on the behavior of the RFP-tagged ATG8i protein and the potential role of cytosolic Ca²⁺ in this process. Osmotic stress was induced with sorbitol and Ca²⁺ accumulation was monitored with a Cameleon transgenic line. Ca²⁺ influx from the apoplast was disrupted using EGTA, and autophagy was analyzed in a transgenic line expressing RFP-ATG8i under its native promoter. We found that osmotic stress triggers Ca²⁺ accumulation, with a pronounced response in the hypocotyl-root transition zone and a weaker response at the root tip. Both osmotic stress and Ca²⁺ signaling promote the accumulation of RFP-ATG8i-labeled autophagosomes in predominantly in hypocotyl-root transition zone. Furthermore, analysis of GFP-HDEL/RFP-ATG8i doubled transgenic line revealed colocalization of RFP-ATG8i with the endoplasmic reticulum marker HDEL, suggesting that ATG8i participates in reticulophagy and may contributed to the ER turnover under stress conditions.

Erv14 is a cargo receptor of COPII vesicles, which is necessary for the efficient trafficking of various membrane proteins. In this work, we demonstrate that the deletion of the ERV14 gene impacts various physiological functions of the vacuole. Compared to the wild‐type cells, cells lacking the ERV14 gene exhibited higher vacuolar pH and altered vacuolar morphology with increased fragmentation. In addition, erv14Δ cells exhibit a thinner cell wall and an impaired process of endocytosis. We also found the importance of ERV14 for cells to overcome environmental stresses, such as neutral external pH, increased zinc and calcium concentrations, and high temperature. Furthermore, comparing gene expression, proteome analysis, and structural modeling revealed new interactions between Erv14 and several vacuolar proteins, including subunits of V‐ATPase and other proteins involved in carbon metabolism.

Ribosome hibernation factors (RHFs) inactivate ribosomes under stress to promote survival but limit biosynthetic capacity. While essential in fluctuating natural environments, this mechanism restricts productivity in laboratory and industrial settings. We found RHFs expression inversely correlates with growth rate and heterologous protein production, revealing a trade-off between stress adaptation and biosynthesis. Our physiological characterization showed broad fitness gains in Δrmf strains across 95 carbon sources, whereas ΔraiA mutants exhibited variable, condition-dependent effects, indicating distinct roles. RHFs promoter activity varied with nutrient availability, displaying transient expression in rich media and sustained activity under poor carbon sources, highlighting ribosomal hibernation’s role in nutrient limitation. Deletion of raiA and rmf in E. coli increased translation, boosting heterologous protein and metabolite yields. Transcriptomics of ΔraiA revealed a shift from stress-responsive to growth-promoting programs. Extending this approach, RHFs inactivation enhanced protein production in Pseudomonas putida and Saccharomyces cerevisiae, establishing ribosomal hibernation as a broadly engineerable target to enhance microbial production.

Autophagy is a highly conserved catabolic process in eukaryotic cells that enables the degradation and recycling of damaged or unnecessary cytoplasmic components. It plays essential roles in both development and responses to environmental stress. In this study, we investigated the regulation of autophagy in response to osmotic stress, focusing on the dynamics of the RFP-tagged ATG8i protein and the potential involvement of cytosolic calcium ion (Ca²⁺) in this process. Our findings indicate that both osmotic stress and Ca²⁺ signaling modulate the accumulation of RFP-ATG8i-labeled autophagosomes in a plant organ-specific manner. Furthermore, the observed colocalization of RFP-ATG8i with the endoplasmic reticulum (ER) marker HDEL suggests a significant role for ATG8i in ER-phagy, highlighting its potential contribution to ER turnover under stress conditions.

Erv14 is a cargo receptor of COPII vesicles, which is necessary for the efficient trafficking of various membrane proteins. In this work, we demonstrate that the deletion of the ERV14 gene impacts various physiological functions of the vacuole. Compared to the wild-type cells, cells lacking the ERV14 gene exhibited higher vacuolar pH and altered vacuolar morphology with increased fragmentation. In addition, erv14Δ cells exhibit thinner cell wall and impaired process of endocytosis. We also found the importance of ERV14 for cells to overcome environmental stresses, such as neutral external pH, increased zinc and calcium concentrations, and high temperature. Furthermore, comparing the gene expression, proteome analysis, and structural modeling revealed new interactions between Erv14 and several vacuolar proteins, including subunits of V-ATPase, and other proteins involved in carbon metabolism.

Legumes associate with Gram-negative soil bacteria called rhizobia, resulting in the formation of a nitrogen-fixing organ, the nodule. Nodules are an important sink for photosynthates for legumes, so these plants have developed a systemic regulation mechanism that controls their optimal number of nodules, the so-called autoregulation of nodulation (AON) pathway, to balance energy costs with the benefits of nitrogen fixation. In addition, soil nitrate inhibits nodulation in a dose-dependent manner, through systemic and local mechanisms. The CLE family of peptides and their receptors are key to tightly controlling these inhibitory responses. In the present study, a functional analysis revealed that PvFER1, PvRALF1, and PvRALF6 act as positive regulators of the nodule number in growth medium containing 0 mM of nitrate but as negative regulators in medium with 2 and 5 mM of nitrate. Furthermore, the effect on nodule number was found to be consistent with changes in the expression levels of genes associated with the AON pathway and with the nitrate-mediated regulation of nodulation (NRN). Collectively, these data suggest that PvFER1, PvRALF1, and PvRALF6 regulate the optimal number of nodules as a function of nitrate availability.

Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological, genomic, and transcriptomic analyses were conducted to obtain insights on molecular responses which could potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H ⁺ , iron, and glycerol transport (i.e. FRE1/2 , JEN1 , VMA2 , VCX1 , KHA1 , AQY3 , and ATO2 ); transcriptional regulation of stress responses to drugs, ROS and heat-shock (i.e. HSF1 , SKN7 , BAS1 , HFI1 , and WAR1 ); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1 , GPA1/2 , RAS2 , IRA2 and REG1 ). At 30°C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results from genomics and transcriptomics revealed that, as part of their adaptive responses, evolved yeast strains aimed to adjust their intracellular pH by H ⁺ and acetic acid transport; modify their metabolism and stress responses via glucose signaling pathways; control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides; and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Furthermore, tolerant strains did not show growth trade off under optimal ancestral conditions. Moreover, the motifs analysis in mutated TFs suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains. This study provides insights into the molecular elements associated with yeast tolerance to acidic pH, acetic acid, and supraoptimal temperatures; information that can be used in inverse metabolic engineering.

The regulation of root Plasma membrane (PM) Intrinsic Protein (PIP)-type aquaporins (AQPs) is potentially important for salinity tolerance. However, the molecular and cellular details underlying this process in halophytes remain unclear. Using free-flow electrophoresis and label-free proteomics, we report that the increased abundance of PIPs at the PM of the halophyte ice plant (Mesembryanthemum crystallinum L.) roots under salinity conditions is regulated by clathrin-coated vesicles (CCV). To understand this regulation, we analyzed several components of the M. crystallinum CCV complexes: clathrin light chain (McCLC) and subunits μ1 and μ2 of the adaptor protein (AP) complex (McAP1μ and McAP2μ). Co-localization analyses revealed the association between McPIP1;4 and McAP2μ and between McPIP2;1 and McAP1μ, observations corroborated by mbSUS assays, suggesting that AQP abundance at the PM is under the control of CCV. The ability of McPIP1;4 and McPIP2;1 to form homo- and hetero-oligomers was tested and confirmed, as well as their activity as water channels. Also, we found increased phosphorylation of McPIP2;1 only at the PM in response to salt stress. Our results indicate root PIPs from halophytes might be regulated through CCV trafficking and phosphorylation, impacting their localization, transport activity, and abundance under salinity conditions.

The regulation of PIP-type aquaporins in the roots of plants has been identified as an important aspect in salinity tolerance. However, the molecular and cellular details underlying this process in halophytes remain unclear. Using free flow electrophoresis and label-free proteomics, we report that the increased abundance of PIPs at the plasma membrane of Mesembryanthemum crystallinum roots under salinity conditions is regulated by clathrin-coated vesicles (CCVs). To understand this regulation, we analyzed several components of the M. crystallinum CCVs complexes: clathrin light chain ( Mc CLC) and subunits μ1 and μ2 of the adaptor complex ( Mc AP1μ and Mc AP2μ). Co-localization analyses revealed the association between Mc PIP1;4 and Mc AP2μ and between Mc PIP2;1 and Mc AP1μ, observations corroborated by mbSUS assays, suggesting that aquaporin abundance at the PM is under the control of CCVs. The ability of Mc PIP1;4 and Mc PIP2;1 to form homo- and hetero-oligomers was tested and confirmed, as well as their activity as water channels. Also, we found increased phosphorylation of Mc PIP2;1 only at plasma membrane in response to salt stress. Our results prompt how root PIPs from halophytes are regulated through CCVs trafficking and phosphorylation, impacting their localization, transport activity and abundance under salinity conditions. One-sentence summary: Abundance of plasma membrane aquaporins in M. crystallinum roots increases in response to salinity via a clathrin-coated vesicle-dependent mechanism.