The role of Atg16 in autophagy, anthocyanin biosynthesis, and programmed cell death in leaves of the lace plant (Aponogeton madagascariensis)

Abstract

Aponogeton madagascariensis, commonly known as the lace plant, produces leaves that form perforations by programmed cell death (PCD). Leaf development is divided into several stages beginning with “pre-perforation” furled leaves enriched with red pigmentation from anthocyanins. The leaf blade is characterized by a series of grids known as areoles bounded by veins. As leaves develop into the “window stage”, anthocyanins recede from the center of the areole towards the vasculature creating a gradient of pigmentation and cell death. Cells in the middle of the areole that lack anthocyanins undergo PCD (PCD cells), while cells that retain anthocyanins (non-PCD cells) maintain homeostasis and persist in the mature leaf. Autophagy has reported roles in survival or PCD promotion across different plant cell types. However, the direct involvement of autophagy in PCD and anthocyanin levels during lace plant leaf development has not been determined. Previous RNA sequencing analysis revealed the upregulation of autophagy-related gene Atg16 transcripts in pre-perforation and window stage leaves, but how Atg16 affects PCD in lace plant leaf development is unknown. In this study, we investigated the levels of Atg16 in lace plant PCD by treating whole plants with either an autophagy promoter rapamycin or inhibitors concanamycin A (ConA) or wortmannin. Following treatments, window and mature stage leaves were harvested and analyzed using microscopy, spectrophotometry, and western blotting. Western blotting showed significantly higher Atg16 levels in rapamycin-treated window leaves, coupled with lower anthocyanin levels. Wortmannin-treated leaves had significantly lower Atg16 protein and higher anthocyanin levels compared to the control. Mature leaves from rapamycin-treated plants generated significantly fewer perforations compared to control, while wortmannin had the opposite effect. However, ConA treatment did not significantly change Atg16 levels, nor the number of perforations compared to the control, but anthocyanin levels did increase significantly in window leaves. We propose autophagy plays a dual role in promoting cell survival in NPCD cells by maintaining optimal anthocyanin levels and mediating a timely cell death in PCD cells in developing lace plant leaves. How autophagy specifically affects anthocyanin levels remained unexplained.

Ac-DEVD-CHO (caspase-3/DEVDase inhibitor) suppresses self-incompatibility–induced programmed cell death in the pollen tubes of petunia (Petunia hybrida E. Vilm.)

“…[ 30 ]). Several main PCD types are currently distinguished in plants, in particular, apoptosis-like PCD, autophagy [ 31 ], aging, and vacuole-mediated cell death or autolysis [ 2 , 32 ]. As for the case of the death of self-incompatible petunia PTs, we may just assume that it is similar to an apoptosis-like PCD according to the morphological traits, such as plasma membrane integrity damage, DNA degradation/fragmentation, and damage of PT structural organization (absence of vacuoles, turgor disturbance, and separation of the cell plasma membrane from the cell wall) [ 19 ].…”

Roadmap for the next decade of plant programmed cell death research

“…However, a role for autophagy in the execution of cell death pathways has also been established, depending on the conditions and cell type. For example, during perforation formation in the lace plant leaves, autophagy plays a dual role in promoting cell survival in non‐PCD cells and mediating timely cell death in PCD cells (Rowarth et al ., 2023). In Arabidopsis root cap, autophagy is involved in the timely cell death of columella cells, but not of the distal root cap cells (Feng et al ., 2022).…”