INTRODUCTION

Rubisco is an abundant protein in plants found within the stromal space of the cell’s chloroplasts. Rubisco is a catalyst for the reaction in which CO₂ forms into a precursor molecule.  This precursor molecule later goes on to be synthesized into carbohydrates.  The formation of the precursor molecule, 3-phosphoplycerate, requires a condensation reaction (Lodish, 2007).

This experiment involved exploring the effect of water availability on the concentration of rubisco.  It has been found that in instances of drought the activity of Rubisco in sugarcane is lessened (Vu, 2009).  Other studies have found that the amount of rubisco within wheat plants such as Triticum aestivum is sensitive to droughts (Demirevska, 2008).  The authors found that under drought conditions, levels of rubisco increased in wheat the C3-metabolic wheat plants.   Since water is needed to complete the 3-phosphoglycerate formation and the activity of the rubisco is lessened in water deficit environments, it is predicted that plants in dry environments will require a higher rubisco concentration in order to insure that the precursor protein to carbohydrate is made. Plants that carry out the C4 photosynthesis mechanism, have an advantage over their C3 counterparts because, C3 plants lose about 97% of their water through transpiration whereas C4 plants loose a much more lower quantity of water, giving them an advantage in dry environments during droughts, high temperatures and nitrogen carbon dioxide limitation. In the C4 pathway in plants, Rubisco is involved in a secondary re-fixation of CO₂ in the bundle sheath cells, where C4 acid decarboxylation provides, at the level of Calvin Cycle, a high CO₂ concentration (da Silva, n.d.). C4 plants spatially concentrates CO₂, unlike CAM plants.

CAM plants grow in more arid environments, where water comes at a premium. CAM plants are able to keep their stomata closed during the hottest and driest part of the day which reduces water loss through evapotranspiration, allowing CAM plants to grow in environments that would otherwise be far too dry. CAM act to concentrate CO₂ around Rubisco, just like C4 plants do, but CAM plants do this in time, providing CO₂ during the day (increasing the efficiency of photosynthesis) and not at night when respiration is the dominant reaction.

 

The type of metabolic pathway may relate to the regulation level of a rubisco.  The regulation of a protein can be controlled in several ways such as the amount of chloropasts, before the protein has been transcribed or after the protein has been transcribed.  Before translation, an organism can inhibit mRNA formation by the use of transcription factors.  A cell can also stop a formed mRNA from undergoing translation.  Post-translation regulation occurs by stopping the protein’s function by dephosphorylation, increasing protease activity, protein blocking factors, etc. (Lodish, 2008).  Rubisco is a protein found in chloroplast which is an organelle with a specific set of DNA.  Regulating a protein after translation has occurred beneficial because the protein is more readily available.  In metabolic pathways in which water is readily available (C4), rubisco would be a limiting factor in producing energy.  Therefore having rubisco regulated after it has been made allows rubisco to be readily available for the metabolic pathway to proceed.  In dry environments, where water availability is the limiting factor for metabolic pathway, rubisco is in greater demand.  Therefore, one would expect rubisco to be regulated by maintaining a high number of chloroplasts in the cell.  If you have a large number of chloroplast organelles, a lot of rubisco can be made quickly.  Subsequently, it would be expected that plants with low water availability would have more chloroplasts so rubisco could be readily available.  However, plants in environments that offer them plenty of water would expect to have less chloroplasts and instead regulate their proteins after they have been made.  This is beneficial because the water-rich plant will not waste energy on maintaining chloroplast if not as many rubisco genes are needed.

 

In this experiment, plant leaves from the following plant species will be used: Sansevieria trifasciata, Pereskia aculeate, Cyprus alternifolius and Hibiscus rosa-sinensis. The Sansevieria trifasciata (also called snake plant of mother-in-law’s tongue) is a drought-tolerant plant found in dry climates in some parts West Africa; it performs the CAM photosynthesis. The Pereskia aculeate (also called the Barbados Gooseberry) is a cactus with leaves, native to the arid climates of tropical South America. The gooseberry plant performs the CAM photosynthesis mechanism. The Cyprus alternifolius (named papyrys) is a plant found in extremely wet environments like Madagascar and it performs the C4 photosynthesis mechanism. Finally, Hibiscus rosa-sinensis (also called the double red hibiscus) is found in moderately wet humid continental environments in which there is seasonal temperature variation like in East Asia. It performs the C4 photosynthetic mechanism. Relating to their various environments, it was hypothesized that the plants in dryer environments will have higher concentrations of rubisco in their leaves. This hypothesis is based on the fact that water is needed to complete the synthesis of carbohydrates and that rubisco is potentially less reactive in dry conditions. Even if the CAM photosynthesis mechanism uses water more efficiently, if rubisco is less active, more will be needed to ensure that enough precursor protein is formed.

The rubisco protein will be measured by isolating the large subunit, which is synthesized in the chloroplast. A western blot analysis will be performed in order to determine relative amounts of rubisco proteins found in the four plant tissues.  The number of genes will be quantified by extracting the DNA and doing a real-time PCR. With the relative rubisco protein concentrations and the number of rubisco genes (or chloroplasts) found in the cell tissue, a comparison between dry and wet plants species can be conducted.  This will relate to the effect of water availability on the concentration of rubisco in plant tissue.  It will be hypothesized that the CAM plants (found in dry environments) will contain more rubisco genes (or chloroplasts) than C4 plants (found in wetter environments).