Candidate Vacuolar Lipases and Vacuolar Processing Enzymes Associated with Late Autophagic Degradation in Carbon-Starved Lupin (Lupinus spp.) Embryonic Axes

The degradation of autophagic bodies in the plant vacuole is required to complete autophagic flux, yet the hydrolases involved in membrane breakdown and cargo processing remain incompletely defined. We investigated candidate vacuolar lipases and vacuolar processing enzymes (VPEs) in embryonic axes of white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet) exposed to carbon starvation. Carbon starvation increased total p-nitrophenyl-palmitate-hydrolyzing and Ac-ESEN-MCA-hydrolyzing activities, whereas asparagine and pharmacological perturbation of autophagic or vacuolar processes attenuated these responses. AlphaFold 3 models, enhanced conformational sampling, and molecular docking indicated that selected candidate lipases possess α/β-hydrolase-like architectures and can accommodate several membrane-associated lipids; however, these predictions do not establish catalytic activity or physiological substrate preference. Vacuole-targeted XP_019420123.1 failed to restore Ape1 maturation or AtAtg8a-GFP processing in Saccharomyces cerevisiae atg15Δ cells, indicating that the construct did not functionally substitute for Atg15 under the conditions tested. By contrast, partial inhibition of VPE-like activity with Ac-YVAD-cmk increased the number of intravacuolar autophagic-body-like structures while phagophores and autophagosomes remained detectable. Transcriptomic and RT-qPCR analyses further revealed gene-specific responses of VPE/legumain transcripts to carbon status and pharmacological treatments. Collectively, the results support an association between VPE-like proteolytic activity and efficient late intravacuolar degradation during carbon starvation. The analyzed lipases remain candidates for vacuolar lipid metabolism, but direct biochemical and genetic evidence is required before assigning them a role in autophagic-body membrane hydrolysis.