Altering abscisic acid biosynthesis to improve water-use efficiency in Populus
Abstract
In an attempt to replace petroleum-based fuels with those from renewable sources, federal and state governing bodies have set specific goals for increased bio-fuel production, resulting in increased demand for resources such as water and arable land. To avoid the displacement of agronomic crops, some potential bioenergy crops will need to be genetically modified to grow on marginal land not generally used for food and feed production. Poplars have received much attention as a potential bioenergy crop due to their fast growth and the ease with which they can be vegetatively propagated. Poplars, however, evolved in an environment where water was abundant and, thus, have low water-use efficiency. Water is perhaps the greatest global constraint to crop growth, and regulation of stomatal function is considered to be a vital adaptive mechanism in the drought response. The enhancement of stomatal closure under water-limiting conditions may lead to plants consuming less water. The LOS5 protein catalyzes the generation of the sulfurylated form of a molybdenum-containing cofactor (MoCo), which is utilized by ABA-aldehyde oxidase in the last step of ABA biosynthesis. To study the effect of LOS5 over-expression, a population of transgenic lines containing the LOS5 gene driven by either the constitutive Super Promoter or the stress-inducible rd29A promoter was generated in the hybrid aspen genotype INRA 717-IB4 (Populus tremula x P. alba). Initially, 71 independent lines were produced and verified for the presence of the selectable marker and gene of interest by standard polymerase chain reaction (PCR). Analysis of LOS5 expression by quantitative real-time PCR (qRT-PCR) was utilized to narrow the population of independent lines down to 20 candidates; consisting of three to four high-, intermediate-, and low-expressers. Analysis of endogenous ABA was performed on these lines using liquid chromatography-mass spectrometry (LC-MS). The highest expresser of LOS5 displayed elevated ABA levels while intermediate- and low- expressers exhibited wild-type levels. Quantitative RT-PCR was used to examine alterations in LOS5, UGT71B6, CYP707A3, and NCED3 expression levels under both osmotic stress and well-watered conditions. No obvious relationship between LOS5 expression level, stress treatment, and expression of UGT71B6, CYP707A3, or NCED3 was observed during these studies. From these results, it may be speculated that a potential threshold level of ABA production exists that must be overcome for endogenous ABA accumulation to overwhelm internal regulatory mechanisms. Analyses of gravimetric water loss and gas-exchange rates were also performed to examine the effect of LOS5 over-expression on transpiration. No transgenic line had gas-exchange or transpiration rates significantly different from wild-type plants. These results agree with previous work performed on Arabidopsis thaliana transformants with elevated levels of endogenous ABA, suggesting that Populus has a similar mechanism for regulating ABA.
Degree
M.S.
Advisors
Meilan, Purdue University.
Subject Area
Plant biology|Forestry|Plant sciences
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