Fig 1.
The lace plant programmed cell death (PCD) system.
(A) Lace plants are grown in axenic Magenta box culture; pre-perforation stage (P), window stage (W), mature stage (M), and imperforate leaves (I). (B) Representative leaves at different stages of development. Imperforate leaves are the first 3–4 leaves to emerge from the corm and do not produce perforations. Pre-perforation stage leaves emerge from the corm with anthocyanin pigmentation. These eventually develop visible areolar “windows” that will eventually become perforated in mature leaves. (C) Details of PCD process. PCD does not take place in imperforate leaves. In pre-perforation leaves, anthocyanin pigmentation is visible, especially at the periphery of the areole (asterisks). PCD can be seen actively occurring in the window stage of development as a gradient of cell death. Non-PCD cells (NPCD, bounded by white dashed lines) persist beyond maturity, early-PCD cells (EPCD, bounded by inner white dashed lines and black dashed lines) have lost their anthocyanins and are destined to die, and late-PCD cells (LPCD, bounded by black dashed lines) are nearly transparent and on the verge of death. Perforation formation is completed in mature stage leaves. Anthocyanin abundance is visibly reduced, and homeostasis for NPCD cells is reached. Scale bars: A = 1 cm, B = 2 cm, C = 70 μm.
Fig 2.
Detection of lace plant Atg16 in lace plant leaves.
RNA and protein were extracted and probed for Atg16 from imperforate (I), pre-perforation (P), window (W), or mature (M) lace plant leaf stages (A-D) or only window and mature leaves from plants treated with either 5 μM rapamycin (Rap), 1 μM wortmannin (Wrt), and 1 μM concanamycin A (ConA) compared to DMSO control (E-H). (A and E) The mean levels of Atg16 mRNA in different lace plant leaf stages were determined by qRT-PCR and normalized to lace plant α-tubulin levels. Protein extracts and a molecular protein standard (L) were resolved by SDS–polyacrylamide gels and blotted to nitrocellulose membranes. (B and F) Membranes and protein lanes were stained with Ponceau-S to serve as loading control before subsequent detection of the presence or absence of ~56 kDa sized Atg16 protein bands with anti-Atg16 antibody (C and G). (D and H) Immunoreactive protein bands were quantitated, and the ratio of Atg16 band intensity to the Ponceau lane signal was averaged. The experiments were performed in triplicate. Means not sharing any letter are significantly different. One-way ANOVA (A, D and H), or Two-way ANOVA, Tukey test (E, P < 0.05; n = 3). Error bars represent the SE.
Fig 3.
Effects of autophagy modulation treatment on mature leaf perforations.
(A) Representative leaves from plants treated with 5 μM rapamycin (Rap), 1 μM wortmannin (Wrt), and 1 μM concanamycin A (ConA) compared to DMSO control. (B) Mean number of perforations formed in mature leaves post-treatment. (C) Mean leaf lengths of mature leaves post-treatment. (B-C) Means not sharing any letter are significantly different. One-way ANOVA, Tukey test (ns = non-significant, P > 0.05, n ≥ 6). Error bars represent the SE. Scale bars = 2 cm.
Fig 4.
Anthocyanin concentration of autophagy modulator treated window or mature stage leaves.
(A) Window stage leaves from plants treated with either DMSO (control), 5 μM rapamycin (Rap), 5 μM wortmannin (Wrt), or 1 μM concanamycin A (ConA) and (B) corresponding micrographs of representative areoles. (C) Anthocyanin extracted from the window and mature leaves from treated plants and (D) absorbance values of window and mature leaves from plants treated with either DMSO (control), 5 μM rapamycin (Rap), 1 μM wortmannin (Wrt), or 1 μM concanamycin A (ConA). Mean anthocyanin concentrations were plotted as standard equivalents of cyaniding-3-rutinoside (C3REs). Means not sharing any individual letters are significantly different. Two-way ANOVA, Tukey test. (ns = non-significant, P > 0.05, n ≥ 3). Error bars represent the SE. Scale bars: A = 2 cm; B = 50 μm.
Fig 5.
Diagram of potential interactions between lace plant PCD, ROS, anthocyanin, autophagy and pertinent genes.
An unknown induction signal(s) during window leaf development initiates the differentiation of PCD from NPCD cells within the areole, visible by an imbalance between reactive oxygen species (ROS) and anthocyanin [16]. RNA-Seq analysis has shown that window leaves up-regulate genes such as Atg18a and SNF1RK that promote autophagy [46]. During window stage of leaf development Atg8 [29] and Atg16 protein levels are high and can be manipulated by autophagy modulators [this study, 56]. PCD cells up-regulate ROS [16] and ROS generating genes peroxidase-4 and primary amine oxidase-1 (PAO-1) while NPCD cells up-regulate anthocyanidin (A3OGT) and UDP flavonoid-3-O-glucosyltransferases (UF3OGT) that promote the production of anthocyanins and a WRKY33 transcription factor that promotes autophagy [46]. Wortmannin (Wrt) and exogenous ROS in the form of H2O2 have similar effects on window stage leaves possibly accentuating cell death and the ROS-anthocyanin gradient imbalance [this study, 16]. On the other hand, rapamycin (Rap) and exogenous antioxidants inhibit anthocyanin accumulation [this study, 16, 48], cell death [29] and perforation formation [This study, 48]. When the ROS-anthocyanin imbalance threshold is not reached this consistently leads to inhibited lace plant PCD [16, 48, 60]. Under normal conditions autophagy can mediate ROS [80] and anthocyanin levels (this study). How autophagic activity, the ROS-anthocyanin ‘gradient’ and associated molecular targets all mediate PCD and intracellular communication between PCD and NPCD cells requires further investigation.