Pitchers of Nepenthes khasiana express several digestive-enzyme encoding genes, harbor mostly fungi and probably evolved through changes in the expression of leaf polarity genes

Background A structural phenomenon seen in certain lineages of angiosperms that has captivated many scholars including Charles Darwin is the evolution of plant carnivory. Evidently, these structural features collectively termed carnivorous syndrome, evolved to aid nutritional acquisition from attracted, captured and digested prey. We now understand why plant carnivory evolved but how carnivorous plants acquired these attributes remains a mystery. In an attempt to understand the evolution of Nepenthes pitcher and to shed more light on its role in prey digestion, we analyzed the transcriptome data of the highly specialized Nepenthes khasiana leaf comprising the leaf base lamina, tendril and the different parts/zones of the pitcher tube viz. digestive zone, waxy zone and lid. Results In total, we generated around 262 million high-quality Illumina reads. Reads were pooled, normalized and de novo assembled to generate a reference transcriptome of about 412,224 transcripts. We then estimated transcript abundance along the N. khasiana leaf by mapping individual reads from each part/zone to the reference transcriptome. Correlation-based hierarchical clustering analysis of 27,208 commonly expressed genes indicated functional relationship and similar cellular processes underlying the development of the leaf base and the pitcher, thereby implying that the Nepenthes pitcher is indeed a modified leaf. From a list of 2386 differentially expressed genes (DEGs), we identified transcripts encoding key enzymes involved in prey digestion and protection against pathogen attack, some of which are expressed at high levels in the digestive zone. Interestingly, many of these enzyme-encoding genes are also expressed in the unopened N. khasiana pitcher. Transcripts showing homology to both bacteria and fungi were also detected; and in the digestive zone, fungi are more predominant as compared to bacteria. Taking cues from histology and scanning electron microscopy (SEM) photomicrographs, we found altered expressions of key regulatory genes involved in leaf development. Of particular interest, the expression of class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIPIII) and ARGONAUTE (AGO) genes were upregulated in the tendril. Conclusions Our findings suggest that N. khasiana pitchers employ a wide range of enzymes for prey digestion and plant defense, harbor microbes and probably evolved through altered expression of leaf polarity genes.

The Evolution of the KANADI Gene Family and Leaf Development in Lycophytes and Ferns

Leaves constitute the main photosynthetic plant organ and even though their importance is not debated, the origin and development of leaves still is. The leaf developmental network has been elucidated for angiosperms, from genes controlling leaf initiation, to leaf polarity and shape. There are four KANADI (KAN) paralogs in Arabidopsis thaliana needed for organ polarity with KAN1 and KAN2 specifying abaxial leaf identity. Yet, studies of this gene lineage outside angiosperms are required to better understand the evolutionary patterns of leaf development and the role of KAN homologs. We studied the evolution of KAN genes across vascular plants and their expression by in situ hybridization in the fern, Equisetum hyemale and the lycophyte Selaginella moellendorffii. Our results show that the expression of KAN genes in leaves is similar between ferns and angiosperms. However, the expression patterns observed in the lycophyte S. moellendorffii are significantly different compared to all other vascular plants, suggesting that the KAN function in leaf polarity is likely only conserved across ferns, gymnosperms, and angiosperms. This study indicates that mechanisms for leaf development are different in lycophytes compared to other vascular plants.

Quantitative analysis of auxin sensing in leaf primordia argues against proposed role in regulating leaf dorsoventrality

Dorsoventrality in leaves has been shown to depend on the pre-patterned expression of KANADI and HD-ZIPIII genes within the plant shoot apical meristem (SAM). However, it has also been proposed that asymmetric auxin levels within initiating leaves help establish leaf polarity, based in part on observations of the DII auxin sensor. By analyzing and quantifying the expression of the R2D2 auxin sensor, we find that there is no obvious asymmetry in auxin levels during Arabidopsis leaf development. We further show that the mDII control sensor also exhibits an asymmetry in expression in developing leaf primordia early on, while it becomes more symmetric at a later developmental stage as reported previously. Together with other recent findings, our results argue against the importance of auxin asymmetry in establishing leaf polarity.

Auxin is not asymmetrically distributed in initiating Arabidopsis leaves

It has been proposed that asymmetric auxin levels within initiating leaves help establish leaf polarity, based in part on observations of the DII auxin sensor. Here we show that the mDII control sensor also exhibits an asymmetry and that according to the ratio-metric auxin sensor R2D2, no obvious asymmetry in auxin exists. Together with other recent findings, our results argue against the importance of auxin asymmetry in establishing leaf polarity.