Abstract
Sugar, the end product of photosynthesis, not only plays an important role in carbon and energy metabolism and in polymer biosynthesis, it also functions as a hormone like signaling molecule that regulates the expression of many genes involved in growth, development and resource allocation in plants. This study focuses on the gene expression profiles in leaves and guard cells of the model plant Arabidopsis thaliana and the response of these genes to sugar. Guard cells are specialized cells that flank each stoma on the surface of the leaf epidermis. Guard cells regulate stomatal aperture thereby controlling the rate of carbon dioxide uptake during photosynthesis and the rate of water loss during transpiration. During stomatal opening, solute content within guard cells builds up due to the uptake of ions and other solutes from outside of the cell. Solute content also builds up by intercellular solute production within guard cells. As a result water is driven into guard cells osmotically, turgor pressure within the cells increases, the guard cells swell, and stomatal aperture increases in size. Stomatal closure is initiated with the dissipation of these solute gradients. Guard cell chloroplasts cannot perform significant amounts of photosynthetic carbon fixation due to a low activity of the enzyme rubisco. Guard cells also lack functional plasmodesmata. Therefore, guard cells depend on extracellular sugars (specifically sucrose). Under the conditions of high light intensity and high transpiration rates, sucrose is swept by the transpiration stream from the mesophyll cells and is deposited in the cell wall of guard cells. Delivery of sucrose into guard cells via sucrose transporters, or breakdown of the sucrose by different sucrose cleaving enzymes on the cell wall and transport of those hexoses into cell, provides the energy needed for guard cell metabolism and may also have a signaling role in the swelling and shrinking of guard cells. This study examines gene expression profiles in leaves, mesophyll cells, and in guard cells, and the transcriptional responses of these genes to sucrose. The ultimate goal of the study was to demonstrate the feasibility of analyzing gene expression in small samples of individually dissected guard cells (about 40-50 guard cells per sample) of Arabidopsis. The panel of genes studied here included several genes preferentially expressed in guard cells (monosaccharide symporter 1 (STP1), trehalose phosphate synthase 1 (TPS1), protein phosphatase 2C (HAB1), potassium inward channel (KAT1) and ADP-glucose pyrophosphorylase (ADPGase)), a gene that should be preferentially expressed in mesophyll cells (ribulose bisphosphate carboxylase, RBCS), two genes inovled in sucrose transport (sucrose transporters 1 and 2, SUC1 and SUC2), and three control genes (Actin-2, ACT2, elongation factor 1a, EF1, and Cyclophilin, CYP). Gene expression was assayed by quantitative real-time PCR (Q-PCR).
In an initial experiment gene expression was analyzed in Arabidopsis leaf strips that were incubated in either 150 mM sucrose or mannitol for 5 hours. This work established the gene expression profile in leaves and showed that the expression of ACT2, HAB1, and KAT1 were up regulated by sucrose, and RBCS was down regulated. RBCS was the most highly expressed of all the genes assayed in leaf strips, followed by SUC1, then STP1, HAB1, and SUC2. CYP, ADPGase, KAT1, and TPS1 were all expressed at low levels in leaf strips. Next, small samples (approximately 10 μg) of mesophyll tissues were microdissected from freeze-dried Arabidopsis leaf strips that had been treated with sucrose or mannitol. RNA was isolated and analyzed by Q-PCR. The mesophyll cells gave a similar profile of gene expression to that observed in the leaf strips. Because of inter sample variation, and because this experiment was done in duplicate rather than triplicate, it was not possible to document sucrose induced changes in gene expression in the mesophyll tissues. Next, the mesophyll RNA samples were diluted 100 and 1000 fold, the RNA was amplified using T7 RNA polymerase, and transcript levels were determined by Q-PCR. The amplified RNA samples gave gene expression profiles that were very similar to that in the unamplified RNA from mesophyll cells. This result indicates the reliability of RNA amplification using T7 RNA polymerase. Finally, samples of guard cells were microdissected from freeze-dried Arabidopsis leaves (50 guard cells per sample), RNA was isolated, amplified using T7 RNA polymerase, and transcript levels were determined by Q-PCR. The gene expression profile observed in the guard cells was quite different from what was found in leaf strips and in mesophyll cells. In guard cells STP1 and KAT1 had the highest level of expression relative to ACT2. TPS1, HAB1, ADPGase, and CYP were all expressed at higher levels in guard cells than in mesophyll cells. SUC2 expression was very low in guard cells, but surprisingly RBCS transcripts levels were moderately high in the guard cell samples. To date very little work has examined changes in gene expression in guard cells because of the difficulty of isolating sufficient numbers of these cells. This study documents the feasibility of studying gene expression in guard cells by manually microdissecting them out of free dried leaves and then amplifiying the RNA with T7 RNA polymerase to give sufficient transcript levels for Q-PCR analysis. This approach opens the possibility of studying the role of gene expression in stomatal movements.
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