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Increasing plants photosynthetic efficienc y is a major challenge that must be addressed in order to cover the food demands of the growing population in the changing climate. Photosynthes i s is greatly limited at the initial carboxylation reaction, where CO2 is converted to the organic acid 3-PGA, catalyzed by the RuBisCO enzyme. RuBisCO has poor affinity for CO2, but also the CO2 concentration at the RuBisCO site is limited by the diffusion of atmospheric CO2 through the various leaf compartments to the reaction site. Beyond genetic engineer-ing, nanotechnology can offer a materials-based approach for enhancing photosynthesis, and yet, it has mostly been explored for the light-dependent reactions. In this work, we developed polyethyleneimine-based nanoparticl e s for enhancing the carbox-ylation reaction. We demonstrate that the nanoparticles can capture CO2 in the form of bicarbonate and increase the CO2 that reacts with the RuBisCO enzyme, enhancing the 3-PGA production in in vitro assays by 20%. The nanoparticles can be introduced to the plant via leaf infiltration and, because of the functionalization with chitosan oligomers, they do not induce any toxic effect to the plant. In the leaves, the nanoparticles localize in the apoplastic space but also spontaneously reach the chloroplasts where photosynthetic activity takes place. Their CO2 loading-dependent fluorescence verifies that, in vivo, they maintain their abi l i t y to capture CO2 and can be therefore reloaded with atmospheric CO2 while in planta. Our results contribute to the development of a nanomaterials-based CO2-concentrating mechanism in plants t h a t can potentially increase photosynthetic efficiency and overall plants CO2 storage., Funding Agencies|European Union [800926, 101042148]; Swedish Research Council [VR-2017-04910]; Swedish Government Strategic Research Area in Materials Science on Advanced Functional Materials at Linkoping University [2009-00971]; MCIN/AEI [Ayuda RYC2021-031668-I]

Leaf senescence can be induced by stress or aging, sometimes in a synergistic manner. It is generally acknowledged that the ability to withstand senescence-inducing conditions can provide plants with stress resilience. Although the signaling and transcriptional networks responsible for a delayed senescence phenotype, often referred to as a functional stay-green trait, have been actively investigated, very little is known about the subsequent metabolic adjustments conferring this aptitude to survival. First, using the individually darkened leaf (IDL) experimental setup, we compared IDLs of wild-type (WT) Arabidopsis (Arabidopsis thaliana) to several stay-green contexts, that is IDLs of two functional stay-green mutant lines, oresara1-2 (ore1-2) and an allele of phytochrome-interacting factor 5 (pif5), as well as to leaves from a WT plant entirely darkened (DP). We provide compelling evidence that arginine and ornithine, which accumulate in all stay-green contexts—likely due to the lack of induction of amino acids (AAs) transport—can delay the progression of senescence by fueling the Krebs cycle or the production of polyamines (PAs). Secondly, we show that the conversion of putrescine to spermidine (SPD) is controlled in an age-dependent manner. Thirdly, we demonstrate that SPD represses senescence via interference with ethylene signaling by stabilizing the ETHYLENE BINDING FACTOR1 and 2 (EBF1/2) complex. Taken together, our results identify arginine and ornithine as central metabolites influencing the stress- and age-dependent progression of leaf senescence. We propose that the regulatory loop between the pace of the AA export and the progression of leaf senescence provides the plant with a mechanism to fine-tune the induction of cell death in leaves, which, if triggered unnecessarily, can impede nutrient remobilization and thus plant growth and survival.

Senescence is regulated by a complex interplay of factors and regulatory circuits, which may be accelerated or delayed depending on the integrated signals. Using a forward genetic screen in Arabidopsis thaliana, we identified a mutant strongly delayed in its induction of senescence in response to prolonged darkness. This mutant, which corresponds to a novel loss-of-function allele of PIF5 (PHYTOCHROME-INTERACTING FACTOR 5), exhibits even slightly more extended survival of leaves in darkness than the previously reported pif5-3 TDNA knock-out line. In the present study, we additionally aimed at deciphering the metabolic and regulatory processes conferring this enhanced capacity for survival in pif5 mutants. We combined physiological, metabolomic and transcriptomic analyses, and discovered that the extended survival of mutant leaves in darkness was associated with reduced protein degradation, slight differences in amino acid catabolism related gene expression as well as strong reduction of amino acid transporter expression, which coincided with enhanced amino acid accumulation. Our findings suggest that enhanced survival in darkness could be mediated by moderate levels of protein degradation allowing build up and slow usage of amino acids as alternative respiratory substrates, while during irreversible senescence, strong degradative processes, together with enhanced amino acid transport either to the site of their metabolization inside the leaf, or to other organs in the plant, could promote the fast progression of senescence and antagonize survival. Comparative metabolomics and gene expression analyses suggested that the senescence regulatory network downstream of PIF5 organizes these irreversible stages of leaf senescence, promoting autophagy and amino acid export, possibly by direct binding of important senescence promoting factors like ORE1 to the promoters of some of the involved genes. The failure to induce these later stages may prolong the reversible phase of d