RNA-Seq analysis reveals potential regulators of programmed cell death and leaf remodelling in lace plant (Aponogeton madagascariensis)

Abstract

Background The lace plant (Aponogeton madagascariensis) is an aquatic monocot that develops leaves with uniquely formed perforations through the use of a developmentally regulated process called programmed cell death (PCD). The process of perforation formation in lace plant leaves is subdivided into several developmental stages: pre-perforation, window, perforation formation, perforation expansion and mature. The first three emerging “imperforate leaves” do not form perforations, while all subsequent leaves form perforations via developmentally regulated PCD. PCD is active in cells called “PCD cells” that do not retain the antioxidant anthocyanin in spaces called areoles framed by the leaf veins of window stage leaves. Cells near the veins called “NPCD cells” retain a red pigmentation from anthocyanin and do not undergo PCD. While the cellular changes that occur during PCD are well studied, the gene expression patterns underlying these changes and driving PCD during leaf morphogenesis are mostly unknown. We sought to characterize differentially expressed genes (DEGs) that mediate lace plant leaf remodelling and PCD. This was achieved performing gene expression analysis using transcriptomics and comparing DEGs among different stages of leaf development, and between NPCD and PCD cells isolated by laser capture microdissection. Results Transcriptomes were sequenced from imperforate, pre-perforation, window, and mature leaf stages, as well as PCD and NPCD cells isolated from window stage leaves. Differential expression analysis of the data revealed distinct gene expression profiles: pre-perforation and window stage leaves were characterized by higher expression of genes involved in anthocyanin biosynthesis, plant proteases, expansins, and autophagy-related genes. Mature and imperforate leaves upregulated genes associated with chlorophyll development, photosynthesis, and negative regulators of PCD. PCD cells were found to have a higher expression of genes involved with ethylene biosynthesis, brassinosteroid biosynthesis, and hydrolase activity whereas NPCD cells possessed higher expression of auxin transport, auxin signalling, aspartyl proteases, cysteine protease, Bag5, and anthocyanin biosynthesis enzymes. Conclusions RNA sequencing was used to generate a de novo transcriptome for A. madagascariensis leaves and revealed numerous DEGs potentially involved in PCD and leaf remodelling. The data generated from this investigation will be useful for future experiments on lace plant leaf development and PCD in planta.

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

“…Both Atg16 mRNA and protein levels were significantly higher in pre-perforation and window stage leaves compared to mature and imperforate leaves. These levels are correlated with early stages of lace plant leaf development when PCD and anthocyanin accumulation is active ( Fig 2A and 2D ) and are also consistent with the RNA-Seq data revealed in Rowarth et al, (2021) [ 46 ]. Pre-perforation and window stage leaves have been shown to up-regulate transcripts encoded for genes involved in autophagy promotion such as Atg16 , Atg18a , and SNF1-related protein kinase KIN10 [ 46 , 55 ] suggesting an increased need for autophagosome formation and cell maintenance during early lace plant developmental PCD.…”

“… 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].…”

“…Transcripts for lace plant Atg16 were used to verify the synthesis results of Atg16 protein RNA sequencing (RNA-Seq) by calculating the relative fold-change in gene expression of samples using the 2 -ΔΔCT method by qRT-PCR [ 46 ]. Equal amounts of RNA (0.1 μg) from treated and control leaf stages were used as a template for cDNA conversion.…”

“…The experiment was performed in triplicate using three biological replicates of imperforate, pre-perforation, window, and mature stage leaves; each preparation was analyzed in duplicate. cDNA copy numbers for Atg16 were determined from a standard curve of Ct values (R 2 > 0.99) and normalized against the lace plant α-tubulin isoform as described and verified in Rowarth et al (2021) [ 46 ].…”

“…The only Atg protein investigated in lace plant development is Atg8 which is detectable in NPCD and PCD cells by using immunostaining but not differentially expressed between cell types and leaf stages [ 29 , 46 ]. Additionally, a transcriptomic analysis of each developmental stage of lace plant leaves revealed that transcripts for Atg16 and Atg18a are highly up-regulated in pre-perforation leaves and window leaves compared to mature leaves and imperforate leaves [ 46 ] while other detectable Atgs genes remained unchanged throughout leaf development. Atg16 has been implicated in the regulation of autophagosome formation in animals and yeast but its specific role in plant autophagy and PCD is not confirmed [ 39 , 47 ].…”