Overview

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant protein in the leaves of light-grown plants.¹ It catalyzes two competing reactions: the photosynthetic CO₂ fixation combines one CO₂ with one ribulose 1,5-bisphosphate (RuBP) to produce two glyceraldehyde-3-phosphate, and in the competing reaction, photorespiratory carbon oxidation takes up O₂ and RuBP to give out CO₂. Rubisco is also an important source of nitrogen recycling and utilization in plants. During leaf senescence, stromal proteins, including Rubisco, are degraded, and the products are transported to growing organs or stored in seeds.

In the higher plants and green algae, Rubisco is comprised of eight small subunits (RBCSs) encoded by a rbcS multigene family in the nucleus and eight large subunits (RBCLs) encoded by a single rbcL gene in the chloroplast.² Genes corresponding to both subunit types are expressed coordinately for the Rubisco holoenzyme synthesis. In Arabidopsis thaliana (At), the small subunits of Rubisco are encoded by four genes divided into the following subfamilies: A (1A) and B (1B, 2B, and 3B).³ Light can influence the transcription and stability of individual RBCS mRNAs, thus affecting organ and specific tissue development.^2,4,5 Coruzzi et al. (1984) showed that under insufficient light, pea leaves turn pale yellow/white, and the expression of RBCS mRNAs is reduced to 1–3% of the expression in green leaves grown in full light.⁴ The pPS-2.4 mRNA accounts for ~30–35% of total RBCS mRNAs in green leaves, whereas it is below the detection level in etiolated leaves. The amount of RBCL protein synthesized is not associated with mRNA levels of other major chloroplast-encoded photosynthesis genes but is correlated with the quantity of RBCS proteins made. RBCS mRNA expressions affect the availability of RBCS proteins, which up-regulate gene expression of RBCL for the Rubisco holoenzyme assembly. The RBCL mRNA levels, and protein synthesis increase in RBCS-sense transgenic rice plants.² A previous study by Izumi et al. (2012) showed that RBCS1A and RBCS3B contribute to the accumulation of Rubisco in Arabidopsis leaves.⁵ These genes work additively to produce enough Rubisco for photosynthesis to occur.⁵ The study demonstrates that total RBCS mRNA determines the amount of Rubisco enzyme in Arabidopsis mature leaves. Different qualities of light also differentially regulate the genes.⁶ For example, unlike AtRBCS3B, the AtRBCS1A gene seems insensitive to blue light.

Quantitative polymerase chain reaction (qPCR) is an extension of traditional PCR. It provides a simple method for determining the amount of a specific sequence or gene in a sample. DNA amplification over time by qPCR is quantitated by measuring fluorescence from dyes intercalated into nascent DNA strands or by the cleavage of target-specific probes. In combination with reverse transcription (RT), RT-qPCR detects and quantifies RNA, and studies the changes in gene expressions, such as measuring the levels of a particular gene expression in different organisms under different developmental stages or treatments.

This lab module investigates the changes in AtRBCS1A and AtRBCS3B mRNA expressions in Arabidopsis thaliana leaves pretreated in the dark for 96 hrs. In Week 1, total RNA is isolated and purified from both the Arabidopsis leaves pretreated in the dark and control leaves without pretreatment, followed by RNA quantification. The integrity of the RNAs is then assessed by formaldehyde agarose gel electrophoresis. In Week 2, the RNAs are amplified by RT-qPCR, the results of which are used to determine the changes in mRNA expressions of AtRBCS1A and AtRBCS3B by the 2^-ΔΔCt method.