Novel Mechanisms Underlying Rubber Accumulation and Programmed Cell Death in Laticiferous Canals of Fruits: Cytological and Transcriptomic Analyses.

Natural rubber is one of the most important industrial raw materials, and its biosynthesis is still a fascinating process that is still largely unknown. In this research, we studied , a unique rubber-producing plant that is different from other rubber-producing species due to the presence of lactiferous canals in its pericarp. The present study aims to provide novel insights into the mechanisms underlying rubber accumulation and PCD by subjecting the laticiferous canals to light microscopy, TUNEL assay, and DAPI staining, as well as viability analysis, cellular ultrastructure analysis, and molecular analysis using light microscopy, scanning electron microscopy, immunofluorescence labeling, transmission electron microscopy, and transcriptome sequencing. At the cellular level, the origin of small rubber particles in the laticiferous canals had no morphological correlation with other organelles, and these particles were freely produced in the cytosol. The volume of the rubber particles increased at the sunken and expanding stage, which were identified as having the characteristics of programmed cell death (PCD); meanwhile, plenty of the rubber precursors or rubber particles were engulfed by the vacuoles, indicating a vacuole-mediated autophagy process. The accumulation of rubber particles occurred after the degeneration of protoplasts, suggesting a close association between rubber biosynthesis and PCD. The molecular analysis revealed the expression patterns of key genes involved in rubber biosynthesis. The upstream genes , and showed a decreasing trend during fruit ripening, while , which is responsible for rubber particle extension, exhibited the highest expression level during the rubber particle formation. Moreover, the transcription factors related to PCD, and showed a negative correlation with the expression pattern of , thus exhibiting strict rules of sequential expression during rubber biosynthesis. Additionally, the expression trends of and , which act as proteases during PCD, were positively correlated with expression. In conclusion, the findings suggest that the autophagic PCD may play a crucial role in rubber accumulation in . Further research is still needed to fully understand the complex regulatory network underlying rubber biosynthesis in plants.