Investigating anthocyanin profile, localization, and transport in Aponogeton madagascariensis
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Lace plant (Aponogeton madagascariensis) forms perforations throughout its leaves via developmental programmed cell death (PCD). Newly emerged ‘pre-perforated’ lace plant leaves are filled with anthocyanin pigmentation, and the first visible sign of cell death is the disappearance of anthocyanins in ‘window’ leaves, which generates a unique PCD gradient. Due to the conspicuous pattern of anthocyanin loss at an early stage, it is evident that anthocyanins play an important role in lace plant PCD. Previous research on anthocyanins in lace plants has identified highly condensed bodies, named anthocyanin vacuolar inclusions (AVIs), that are visible in the apex of window leaves using light microscopy, however, the structure of AVIs and their function in leaf development and PCD remains unclear. Further, the intracellular transport of anthocyanins from the endoplasmic reticulum (ER; biosynthesis site) to the vacuole (accumulation site) is unknown in lace plant. Three models have been proposed: vesicular, ligandin, or autophagic transport. Even though the involvement of the ER is well-documented in animal PCD, little is known about ER dynamics in lace plant PCD. Therefore, the objectives of the study are (1) to isolate and identify anthocyanin species present in different lace plant tissues, (2) to investigate anthocyanins and AVIs in lace plant leaves by (i) determining the ultrastructure and localization of anthocyanins and AVIs, (ii) determining the mode of intracellular transport of anthocyanins, and (iii) investigating ER changes across the PCD gradient, and (3) to determine the role of anthocyanins in lace plant PCD. Ultra-pressure liquid-chromatography (UPLC) and mass-spectrometry (MS) were utilized to profile anthocyanins in lace plant leaves and inflorescences. A combination of long-term, live-cell imaging and transmission electron microscopy (TEM) were used to determine the localization of AVIs in lace plant leaves. A protocol was optimized for 3,3'-dihexyloxacarbocyanine iodide, a fluorescent dye, to observe ER changes in lace plant leaves across the PCD gradient using confocal laser scanning microscopy (CLSM). TEM was also used to compare ER in healthy and dying cells. Lastly, pharmacological experimentation was performed with anthocyanin modulators, methyl jasmonate (a biosynthesis promoter) and phenidone (a biosynthesis inhibitor), to determine the effect on perforation formation in lace plant leaves. Results indicated that anthocyanin profiles differ among the various developmental leaf stages and inflorescences. Microscopic observations determined that AVIs are more visible in the apex of window leaves compared to the mid-regions. Based on CLSM results, two forms of anthocyanin bodies that differed at the leaf apex and mid-regions were observed: a large, singular spherical body (approximately 1-2 µm in size) located in cells at the window apex and multiple small, spherical bodies (less than 1 µm) located in vasculature and NPCD cells in the mid-regions. Transmission electron microscopy of areoles in the mid-regions of window stage leaves revealed that AVIs are deposited in ER-derived vesicles for transportation to the vacuole, which supports the vesicular transport model. CLSM provided evidence of ER changes between developmental leaf-stage, as well as across the PCD gradient. Addition of anthocyanin modulators significantly increased (methyl jasmonate treatment) and decreased (phenidone treatment) anthocyanin content in lace plant leaves, but the anthocyanin modulators did not significantly affect perforation number at the concentrations used. The results of the study suggest the differential occurrence of anthocyanin species and their potential role at specific stages of lace plant leaf development and contributes to the understanding of AVIs and anthocyanin transport, as well as corresponding structural changes taking place in the ER, an organelle involved in the transport mechanism of anthocyanins.