Profiling Phenolic Compounds in Aponogeton madagascariensis and Investigating Their Role in Programmed Cell Death During Leaf Development

Abstract

The lace plant (Aponogeton madagascariensis) is an aquatic monocot native to Madagascar that undergoes a form of programmed cell death (PCD) during early leaf development. This process results in a perforated leaf morphology that is extremely rare among plants. Young leaves emerge with a strong red pigmentation due to the presence of anthocyanins, a family of phenolic compounds. As lace plant leaves develop, they form a predictable pattern of perforations throughout the leaf lamina, starting in the centre of areoles located in between the lateral and longitudinal veins. The first sign of this PCD process is the disappearance of anthocyanin pigmentation in the young window stage leaf. Phenolic compounds are ubiquitous among plant life, and are known to be involved in a variety of plant stress responses and defense mechanisms. The production and regulation of phenolics are known to be heavily linked with jasmonates, a small family of phytohormones. Although the mechanisms of phenolic involvement in stress response are widely understood, their role in plant PCD remains to be elucidated. In this study, a profiling of anthocyanin species and other phenolics was conducted in key developmental stages of lace plant leaf development via microscopy, spectrophotometry, and mass spectrometry. In vivo pharmacological experiments were conducted on whole plants by treatments with vanillin, phenidone, and methyl jasmonate to quantify their effects on leaf perforation formation. Results indicate that anthocyanin and vanillin content were significantly higher in young leaves prior to and during PCD. In addition, anthocyanins are heavily localized in the apices of lace plant leaves. Under stresses such as high pH, light deprivation, and crowded growth, the apex of the leaf is the location of the formation of membrane-less anthocyanic vacuolar inclusions (AVIs). Treatment with inhibitors of endogenous phenolics through the jasmonate pathway, phenidone and vanillin, showed significant reduction of anthocyanin, total phenolics, DPPH radical scavenging capacity, and PCD in the lace plant. Treatment with methyl jasmonate (MeJA) caused a significant increase in anthocyanin production and DPPH radical scavenging, but did not affect the number of perforations. Analyses by Triple Quadrupole Mass Spectrometry analyses allowed for the identification of anthocyanin species present in the lace plant for the first time. Relative abundance analyses from TQ-MS revealed a substantial decrease in delphinidin species during leaf development after the PCD process is complete. Additionally, these results also revealed potentially novel compounds that have not been documented in any other species to date. The effects of plant phenolics on PCD mechanisms could shed light on future applications in regulating cell death for pharmacological and agricultural research.