lia chaer/uspWater from the tank feeds the plant and shelters animalslia chaer/usp
The Vriesea gigantea bromeliads live on treetops and gather water between their leaves – this is why they are called tank epiphytes. It is a common belief that plants of this type are the only plants that prefer to extract nitrogen directly from urea, which is found abundantly in the urine of frogs that use puddles of water for shelter and to lay their eggs. The group coordinated by botanist Helenice Mercier, from the University of São Paulo/USP discovered recently that these bromeliads have two strategies to capture urea and found unique physiological mechanisms.
Seeking to find more details in Helenice’s findings – that tank bromeliads use urea – Cassia Takahashi chopped up many vriesea leaves to examine in detail how they absorb nitrogen, the chemical element that is necessary to build proteins for growth and reproduction. She used a microscope to observe in detail that the base of each leaf has a higher density of trichomes, fine hairs that act like miniature roots. The tips of the leaves have 70% of the number of trichomes found on the base, double the number of stomas, the structures that open and close to allow breathing and photosynthesis, according to the article published in the Brazilian Journal of Plant Physiology in 2007.
The external morphology indicates that the base of the leaves functions like a root, absorbing water and nutrients and the tips function like leaves themselves, where most of the photosynthesis occurs. But Cassia was interested in the physiology, in what happens inside the plant that allows it to absorb this organic nitrogen. She had to find and quantify the enzymes responsible for the processing of the urea and the by-products. When this substance found in animal urine enters the plant, the urease enzyme breaks the urine into ammonia and carbon gas (CO2). Then the other enzymes go into action, especially glutamate synthetase (SG), which have a great attraction for ammonia, and integrate it into the glutamine amino acid. In the beginning, the young botanist concentrated her efforts on the base of the leaves, which remains under water where the nutrients are found, and threw away everything else. This seemed very wasteful. “I decided to see if the leaf was all the same, to find out if it would be possible to use all of it and I discovered that I was looking in the wrong place”, she says. Most of the synthetase glutamine was stored in the tips of the leaves, proving that the nitrogen is actually assimilated there. This discovery gave the project a new impetus. “It was the first time that a functional, physiological function was shown in a leaf”, says Helenice. This organization makes sense: the nitrogen is assimilated where photosynthesis happens and excess energy exists to feed this process and produce proteins. The result also suggests that the ammonia is transported from the base to the tip of the leaves, which was also unexpected. “The ammonia is a very toxic substance that plants usually assimilate in organic molecules as soon as they absorb it”, Helenice explains. When highly concentrated, the ammonia can block the plants’ respiratory chain, a serious problem in leaves, which are responsible for breathing and photosynthesis. She still doesn’t know how the Vriesea gigantea avoids these problems.
miguel boyayanSterile nursery: in the lab, bromeliads grow in a bacteria-less environmentmiguel boyayan
Controlled environment
One of the problems that the researchers found was that the bacteria, natural inhabitants of the bromeliads’ tanks, also need nitrogen, and transform it into different compounds. This is why it is necessary to cultivate the plants in sterilized flasks. The group also discovered that it is necessary to know the age of the plants. At the age of two, the bromeliads of this species are approximately two centimeters tall and the leaves grow wider and form the tank at this point. At this moment, the distribution of the trychomes and the physiology are modified, and therefore mixing plants of different ages in the same experiment leads to errors. In addition, in order to maintain control over the intake of nitrogen, it necessary to keep frogs out of the greenhouse. The same holds true for spiders, whose feces are also a source of nitrogen for the bromeliads. In 2006, ecologist Gustavo Romero, from the Paulista State University/Unesp, in the city of São José do Rio Preto, showed that 18% of the nitrogen consumed by the Bromeliad balansae species comes from the Psecas chapoda spider. This is why the team from USP tries to keep its plants impeccably. At one point, Cassia washed each leaf, one by one, of hundreds of bromeliads, using a toothbrush to deal with a pest. In the lab, the bromeliads are cultivated in a sterile environment that excludes bacteria.
In nature, the water accumulated in the bromeliad is a scenario of the competition between plant and bacteria. In an article published in New Phytologist in 2007, Austria’s Erich Inselsbacher, who spent part of his master’s program at the USP lab, showed that the vriesea have two ways of benefiting from the urine of the amphibians. In the presence of urea, the plant releases urease into the tank, absorbs the ammonia produced by the reaction, which is then processed by the internal enzymes. “The plant or bacteria with the most hunger for ammonia is the winner”, says Helenice. The vriesea’s synthetase glutamine is avid, but the visitor from Austria showed that the bromeliad also absorbs whole urea much more efficiently than the other nitrogen substances.
Plants normally have a point of saturation in their capacity to absorb substances, but this does not happen with urea. The more urea the researchers put inside the tank, the more urea the V. gigantea absorbed. This observation led the team from USP to infer the existence of a protein pore specialized in absorbing the urine’s nitrogen compound. During her doctorate studies, Camila Cambuí looked for a candidate among the genes of the aquaparins, proteins that form the pores for the entry of water and other substances. In collaboration with botanist Marília Gaspar, from the Botanical Institute of São Paulo, she found the urea’s entry into the vriesea, and is now trying to produce a new aquaporin in the lab to conduct experiments and see in detail how it functions. For the time being, part of the mystery has been unveiled: “All of the urea apparently enters the leaf and is directly assimilated; in addition, the urea can be broken up into ammonia”, Helenice explains.
miguel boyayanFertile ground: the leaves of the Vriesea gigantea are full of surprisesmiguel boyayan
In her opinion, this aquaporin of the vrísea probably appeared in the bromeliads that co-evolved with the amphibians that deposit eggs and urine there. The plants also developed strategies to use this source of nutrition to the highest extent; when the bromeliad tank gets a dose of urea, in the first 24 hours the plant gives preference to this substance, even if other sources of nitrogen, such as ammonia or nitrate, are present. This preference is important because urea is an inconsistent resource; frogs look for the bromeliads’ tanks when it rains more and lots of water accumulates. At this time, the plant needs to absorb as much as possible, competing with the bacteria. Plant debris and decomposing insects are the sources of nitrogen during dry periods.
In an article to be published shortly in Physiologia Plantarum journal, Camila specified more details on how urea is processed when it enters the plant: 40% of the activity of the urease from the V. gigantea takes place outside the cells, on the cell walls and membranes, unlike the tankless species, where the urease is concentrated inside them. In addition to being high in nitrogen, the processed urea also triggers carbon gas. Camila showed that this carbon immediately aggregates itself to the chloroplasts, where photosynthesis takes place and is soon used to build up cellulose, which makes the plant grow. This is why the tank bromeliads fertilized with urea grow faster – something that commercial growers of these plants had already realized. They know, for example, that it is enough to place urea in the tanks of the bromeliads, instead of on the leaves and the roots, to reduce the effects of the exaggerated use of nitrogen in commercially cultivated plants, In partnership with Helenice, Gustavo Romero is now trying to prove that the urea absorbed by bromeliads in the wild actually comes from the urine of frogs.
Scientific articles
CAMBUÍ, C.A. et al. Detection of urease in the cell wall and membranes from leaf tissues of bromeliad species. Physiologia Plantarum. In print.
INSELSBACHER, E. et al. Microbial activities and foliar uptake of nitrogen in the epiphytic bromeliad Vriesea gigantea. New Phytologist. v. 175, n. 2, p. 311-320. Jul. 2007.
TAKAHASHI, C.A. et al. Differential capacity of nitrogen assimilation between apical and basal leaf portions of a tank epiphytic bromeliad. Brazilian Journal of Plant Physiology. v. 19, n. 2, p. 119-126. Apr.-Jun. 2007.
