Alternative oxidase (AOX) in the carnivorous pitcher plants of the genus Nepenthes: what is it good for?

BACKGROUND AND AIMS: The carnivorous pitcher plants of the genus Nepenthes have evolved modified leaves that act as pitcher traps. The traps are specialized for prey attraction, capture, digestion and nutrient uptake but not for photosynthetic assimilation. METHODS: In this study, we used antibodies against different photosynthetic (D1, Lhcb2, Lhcb4, RbcL) and respiratory-related (AOX, COXII) proteins for semi-quantification of these proteins in the assimilation part of the leaves and the pitcher traps of different Nepenthes species and hybrids. Different functional zones of the trap and the traps from different ontogenetic stages were investigated. The pitcher traps of the distantly related species Sarracenia purpurea ssp. venosa were used as an outgroup. In addition, chlorophyll fluorescence and infrared gas analysis were used for measurements of the net rate of photosynthesis (AN) and respiration in the dark (RD). KEY RESULTS: The pitcher traps contained the same or lower abundance of photosynthesis-related proteins in accordance with their low AN in comparison to the assimilation part of the leaves. Surprisingly, all traps contained a high amount of alternative oxidase (AOX) and low amount of cytochrome c oxidase subunit II (COX II) than in the assimilation part of the leaves. Thermal imaging did not confirm the role of AOX in pitcher thermogenesis. CONCLUSIONS: The pitcher traps contain a high amount of AOX enzyme. The possible role of AOX in specialized pitcher tissue is discussed based on knowledge of the role and function of AOX in non-carnivorous plants. The roles of AOX in prey attraction, balance between light and dark reactions of photosynthesis, homeostasis of reactive oxygen species, digestive physiology and nutrient assimilation are discussed.

Enzyme activities in two sister-species of carnivorous pitcher plants (Nepenthes) with contrasting nutrient sequestration strategies.

The carnivorous pitcher plants of the genus Nepenthes usually attract, capture and digest arthropod prey to obtain mineral nutrients. But few members of the genus have evolved specialized nutrient sequestration strategies to acquire nitrogen from the faeces and urine of mutualistic mammals, which they attract. Because the plants obtain significant amounts of nitrogen in a more available form, we hypothesized that they have relaxed the production of digestive enzymes. If so, species that digest mammal faeces should show fewer digestive enzymes than closely related species that rely on arthropods. We tested this hypothesis by comparing digestive enzymes in 1) Nepenthes hemsleyana, whose pitchers serve as roosts for the mutualistic woolly bat Kerivoula hardwickii, which also defecate inside the pitchers, and 2) the close relative Nepenthes rafflesiana, a typical arthropod capturing species. To investigate the dynamics of aspartic proteases (nepenthesin I and II) and type III and IV chitinases in both species, we conducted qPCR, western blotting, mass spectrometry, and enzyme activity measurements. We found that mRNA in pitcher tissue and enzyme abundance in the digestive fluid is upregulated in both species in response to faeces and insect feeding. Contrary to our initial hypothesis, the final nepenthesin proteolytic activity in the digestive fluid is higher in response to faeces addition than to insect prey irrespective of Nepenthes species. This indicates that faeces can mimic arthropod prey triggering the production of digestive enzymes and N. hemsleyana retained capacity for production of them.

Jasmonate-independent regulation of digestive enzyme activity in the carnivorous butterwort Pinguicula × Tina.

Carnivorous plants within the order Caryophyllales use jasmonates, a class of phytohormone, in the regulation of digestive enzyme activities. We used the carnivorous butterwort Pinguicula × Tina from the order Lamiales to investigate whether jasmonate signaling is a universal and ubiquitous signaling pathway that exists outside the order Caryophyllales. We measured the electrical signals, enzyme activities, and phytohormone tissue levels in response to prey capture. Mass spectrometry was used to identify proteins in the digestive secretion. We identified eight enzymes in the digestive secretion, many of which were previously found in other genera of carnivorous plants. Among them, alpha-amylase is unique in carnivorous plants. Enzymatic activities increased in response to prey capture; however, the tissue content of jasmonic acid and its isoleucine conjugate remained rather low in contrast to the jasmonate response to wounding. Enzyme activities did not increase in response to the exogenous application of jasmonic acid or coronatine. Whereas similar digestive enzymes were co-opted from plant defense mechanisms among carnivorous plants, the mode of their regulation differs. The butterwort has not co-opted jasmonate signaling for the induction of enzyme activities in response to prey capture. Moreover, the presence of alpha-amylase in digestive fluid of P. × Tina, which has not been found in other genera of carnivorous plants, might indicate that non-defense-related genes have also been co-opted for carnivory.