Transcriptomic and metabolomic responses to varying nutrient conditions reveal new insights into pitcher formation in Nepenthes khasiana.

Nepenthes are carnivorous plants that colonize habitats poor in soil nutrients. To survive, Nepenthes develop pitchers capable of capturing and digesting attracted prey. Prey-derived nutrients are then absorbed to support plant growth and reproduction. So far, pitcher formation in Nepenthes is a poorly understood biological process. To shed light on the formation of Nepenthes pitchers, we grew dissected shoot apices of 3-month-old N. khasiana seedlings in Murashige and Skoog (MS) medium of varying strengths viz. full-strength MS (1 MS), quarter-strength MS (1/4 MS), and one-eighth strength MS (1/8 MS), including those lacking nitrogen (N), phosphorus (P), and potassium (K) and in the presence of N-1-naphthylphthalamic acid (NPA). We sequenced the transcriptome and performed gas chromatography-mass spectrometry to determine changes in gene expression patterns and primary metabolite accumulations in response to the varying nutrient conditions. Shoots grown in 1 MS or NPA-containing 1/4 MS and 1/8 MS failed to develop pitchers. Remarkably, pitcher formation is restored when N was removed from 1 MS. Transcriptomic response to nutrient-sufficient and nutrient-deficient conditions are associated with the enrichment of several defence-related genes, including two JA-mediated defence response genes, WRKY51 and WRKY11, respectively. Further, metabolomic response to the varying nutrient conditions identifies glutamic acid as a key metabolite, accumulating at lower and higher levels in shoots with and without pitchers, respectively. Together, our findings suggest that failure to form pitchers may be associated with the suppression of the JA-signalling pathway, whereas the induction of the JA-mediated defence response is linked to pitcher formation in N. khasiana.

Genetic Basis of Carnivorous Leaf Development.

Plant carnivory is often manifested as dramatic changes in the structure and morphology of the leaf. These changes appear to begin early in leaf development. For example, the development of the Sarracenia purpurea leaf primordium is associated with the formation of an adaxial ridge, whose growth along with that of the leaf margin resulted in a hollow structure that later developed into a pitcher. In Nepenthes khasiana, pitcher formation occurs during the initial stages of leaf development, although this has not been shown at the primordial stage. The formation of the Utricularia gibba trap resulted from the growth of the dome-shaped primordium in both the longitudinal and transverse directions. Recent research has begun to unfold the genetic basis of the development of the carnivorous leaf. We review these findings and discuss them in relation to the flat-shaped leaves of the model plant Arabidopsis.

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.

ASYMMETRIC LEAVES1 and REVOLUTA are the key regulatory genes associated with pitcher development in Nepenthes khasiana.

Nepenthes develops highly specialized insect-eating organs called pitchers that provide adequate insect-derived nutrients to the plants to offset low nutrient availability in their natural habitat. But so far, the molecular basis of Nepenthes pitcher development remains largely unknown. In an attempt to unravel the underlying mechanisms of pitcher formation, we made morphological observations of the developing N. khasiana leaf and performed RNA-seq to identify genes controlling pitcher development. Histology and scanning electron microscopy photomicrographs show that pitcher formation in N. khasiana occurs early in development and shares anatomical features with the young in-rolled leaf base lamina. Analysis of the RNA-seq data indicated that the modification of the leaf into a pitcher is associated with the altered expressions of leaf polarity genes ASYMMETRIC LEAVES1 (AS1) and REVOLUTA (REV). In fact, both genes displayed exclusive or relatively higher expressions in the tip of the leaf that later developed into a pitcher. We propose that NkAS1 may act to inhibit lamina outgrowth and promote the formation of the tendril. Increased NkREV expression may have been involved in the formation of the N. khasiana pitcher. This dataset will allow further research into this area and serve as the basis for understanding Nepenthes pitcher development.

Metabolic shift in sugars and amino acids regulates sprouting in Saffron corm.

Saffron is one of the most expensive spices of the world. Since this spice is triploid and meiosis is unusual, it cannot reproduce sexually like other plants; rather, it is propagated vegetatively via an underground corm, which can withstand a long dry dormant period before sprouting. Thus, corms are indispensable to saffron propagation. To identify and analyse signature metabolites associated with the 'dormancy-sprouting' process, non-targeted GC-MS was performed at different stages of corm development. Comparative metabolite profiling reflected dissimilar profiles among the stages as portrayed by differential cluster patterns of metabolites in the PCA and PLS-DA analysis. Correlation analysis revealed the interdependencies of individual metabolites and metabolic pathway. At the onset of stage 2, characterized by the initiation and differentiation of leaf primordia, a shift from dormancy to active metabolism occurred as derived from the increased abundance of sugars and other metabolites involved in the tricarboxylic acid cycle, glycolytic, amino acid and fatty acid pathways. These changes contribute to sprouting and vegetative growth of the corm. The present study provides new insights into saffron corm composition and metabolite changes associated with various stages of corm development and may pave the way for achieving agronomical improvements in this economically important spice.

What determines a leaf's shape?

The independent origin and evolution of leaves as small, simple microphylls or larger, more complex megaphylls in plants has shaped and influenced the natural composition of the environment. Significant contributions have come from megaphyllous leaves, characterized usually as flat, thin lamina entrenched with photosynthetic organelles and stomata, which serve as the basis of primary productivity. During the course of evolution, the megaphylls have attained complexity not only in size or venation patterns but also in shape. This has fascinated scientists worldwide, and research has progressed tremendously in understanding the concept of leaf shape determination. Here, we review these studies and discuss the various factors that contributed towards shaping the leaf; initiated as a small bulge on the periphery of the shoot apical meristem (SAM) followed by asymmetric outgrowth, expansion and maturation until final shape is achieved. We found that the underlying factors governing these processes are inherently genetic: PIN1 and KNOX1 are indicators of leaf initiation, HD-ZIPIII, KANADI, and YABBY specify leaf outgrowth while ANGUSTIFOLIA3 and GROWTH-REGULATING FACTOR5 control leaf expansion and maturation; besides, recent research has identified new players such as APUM23, known to specify leaf polarity. In addition to genetic control, environmental factors also play an important role during the final adjustment of leaf shape. This immense amount of information available will serve as the basis for studying and understanding innovative leaf morphologies viz. the pitchers of the carnivorous plant Nepenthes which have evolved to provide additional support to the plant survival in its nutrient-deficient habitat. In hindsight, formation of the pitcher tube in Nepenthes might involve the recruitment of similar genetic mechanisms that occur during sympetaly in Petunia.

Assessment of phylogenetic inter-relationships in the genus Cymbidium (Orchidaceae) based on internal transcribed spacer region of rDNA.

Sequence data obtained from nrITS region were used to assess phylogenetic inter-relationships and infrageneric classification of ten Cymbidium species collected from north-east India. The final aligned data matrix of combined ITS 1, 5.8S and ITS 2 yielded 684 characters. The ITS 1 and ITS 2 regions showed variable sequence lengths and G+C content (%). The 5.8S region was found to be more conserved (98.71%) followed by ITS 1 (86.12%) and ITS 2 (69.40%). ITS 2 recorded highest percentage of parsimony informative sites (7.46%), high sequence divergence with indels (24.63%), high number of transitions and transversions. ITS sequence data determined the phylogeny of Asiatic Cymbidiums with high bootstrap values. All three proposed subgenera could be distinguished clearly by all four (MP, ML, NJ, and BI) phylogenetic methods. This study validates the utility of ITS rDNA region as a reliable indicator of phylogenetic relationships, especially ITS 2 as probable DNA barcode at higher levels and can serve as an additional approach for identification of broader range of plant taxa especially orchids.