Prone to loss: Senescence-regulated protein degradation leads to lower protein extractability in aging tomato leaves.

The utilization of proteins extracted from tomato (Solanum lycopersicum) leaves as cost-effective resources for human consumption or animal feed has gained interest. Thus, increasing protein extractability from tomato leaves became a new breeding target. However, the genetic factors influencing this trait remains poorly understood. In this study, we analyzed changes in leaf protein content, protein composition, and extraction yield across developmental stages, which are vegetative growth, flowering, fruit-forming, and mature fruit. Moreover, tomato gene expression across developmental stages was also studied, to identify genes underlying variability in leaf protein extraction. Protein extraction yield decreased from 0.51 g/g to 0.01 g/g leaf protein from the vegetative to mature stage. However, total protein content inferred with Dumas combustion analysis did not change over the developmental stages tested, while the protein-to-peptide ratio decreased significantly. To further analyze potential causes underlying the decline of protein-to-peptide ratio, the enzymatic activity of proteases - i.e. the enzymes responsible for protein degradation - and the expression of genes encoding these enzymes was studied along plant development. The overall specific activity of proteases did not change significantly throughout plant development. On the contrary, the gene expression of distinct members of the aspartic, cysteine, and subtilase protease families increased. Overall, our findings suggest that extraplastidic protein degradation likely underlies the protein degradation observed during senescence. In the future, the reduction of the activity of extraplastidic proteases through biotechnology could represent an effective strategy to develop tomato varieties with improved protein extraction yields.