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Plants were then transferred to 6-inch diameter pots and cultivated at the University of Calgary greenhouse at 23 ± 3 °C with a 16-h photoperiod.
Fine powder (100 mg) ground in liquid nitrogen from various tissues or fresh latex (100 mg) was immediately mixed with 1 ml of TRIzol reagent (Invitrogen).
Classical biochemical studies using latex have proposed a model where NR is synthesized on a monolayer organelle, known as the rubber particle, suspended in the latex (19).
A rubber synthase complex is proposed to reside on the membrane of rubber particles with its catalytic domain exposed to the cytosol.
The rubber particles were resuspended in latex storage buffer.
In this model, the newly synthesized NR polymer extends into the core of the rubber particle using IPP substrates from the cytoplasm.
However, molecular evidence for this model is scarce, and the rubber synthase mystery has remained unsolved for the past 50 years.
It is important to note that 2,500 plant species can produce NR with g/mol (17, 18).
Therefore, studies of NR from these Asteraceae plants can help elucidate NR biosynthesis in plants.