Activation of stress-response genes by retrograde signaling-mediated destabilization of nuclear importin IMPα-9 and its interactor TPR2.

Deciphering stress-induced retrograde signal transmission from plastids to the nucleus has long puzzled plant biologists. To address this, we performed a suppressor screen of the ceh1 mutant, known for elevated MEcPP levels, and identified the gain-of-function mutant impα-9, which reverses dwarfism and suppresses stress-response genes in the ceh1 background despite heightened MEcPP. Subsequent genetics and biochemical analyses established that the accumulation of MEcPP initiates an upsurge in ASK1 abundance, a pivotal component in the proteasome degradation pathway. This increase in ASK1 prompts the degradation of IMPα-9. Additionally, we uncovered a protein interaction between IMPα-9 and TPR2, a transcriptional co-suppressor. Reduction in IMPα-9 levels coincides with a decrease in TPR2 abundance. Significantly, these interactions were disrupted in impα-9 mutants, highlighting the critical role of a single amino acid alteration in maintaining these associations. Disruption of these interactions results in the reversal of MEcPP-associated phenotypes. ChIP-seq analyses unveiled TPR2's binding to stress response genes and suggested IMPα-9-DNA association. Together, these associations function to suppress stress genes under normal conditions, but this suppression is alleviated in response to stress through the degradation of the suppressing machinery. The biological relevance of these findings was emphasized during high light stress, characterized by MEcPP accumulation, elevated ASK1 levels, degradation of IMPα-9, reduced TPR2 abundance, and subsequent activation of a network of stress response genes. In essence, our study uncovers new insights into plant adaptive responses, revealing complex interactions among retrograde signaling, the proteasome, and nuclear transport machinery, and establishes plastids as a regulatory stress response hub.

Ice plant root plasma membrane aquaporins are regulated by clathrin-coated vesicles in response to salt stress.

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.

A differential subcellular localization of two copper transporters from the COPT family suggests distinct roles in copper homeostasis in Physcomitrium patens.

The moss Physcomitrium (Physcomitrella) patens is a bryophyte that provides genetic information about the adaptation to the life on land by early Embryophytes and is a reference organism for comparative evolutionary studies in plants. Copper is an essential micronutrient for every living organism, its transport across the plasma membrane is achieved by the copper transport protein family COPT/CTR. Two genes related to the COPT family were identified in Physcomitrella patens, PpaCOPT1 and PpaCOPT2. Homology modelling of both proteins showed the presence of three putative transmembrane domains (TMD) and the Mx3M motif, constituting a potential Cu + selectivity filter present in other members of this family. Functional characterization of PpaCOPT1 and PpaCOPT2 in the yeast mutant ctr1Δctr3Δ restored its growth on medium with non-fermentable carbon sources at micromolar Cu concentrations, providing support that these two moss proteins function as high affinity Cu + transporters. Localization of PpaCOPT1 and PpaCOPT2 in yeast cells was observed at the tonoplast and plasma membrane, respectively. The heterologous expression of PpaCOPT2 in tobacco epidermal cells co-localized with the plasma membrane marker. Finally, only PpaCOPT1 was expressed in seven-day old protonema and was influenced by extracellular copper levels. This evidence suggests different roles of PpaCOPT1 and PpaCOPT2 in copper homeostasis in Physcomitrella patens.