Identification and expression of invertase genes in Populus.

摘要

Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible hydrolysis of sucrose into glucose and fructose. These sugars can act as both metabolic fuel and as signaling compounds directly affecting resource allocation in the plant and indirectly influencing the expression of genes responsive to shifts in hexose and sucrose availability. The invertase family in plants is composed of two sub-families thought to have distinct evolutionary origins and can be distinguished by their pH optima for activity: acid invertases and neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in cyanobacteria and function in the cytosol. The recently sequenced genome of Populus trichocharpa (Torr. & Gray) allowed us to identify the genes encoding invertase in this woody perennial. Here we describe the identification of eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1-3), and five of which belong to the cell wall targeted group (PtCIN1-5). Similarly, we report the identification of 16 neutral/alkaline invertase genes (PtNIN1-16). Expression analyses using whole genome microarrays and RT-PCR reveal evidence for expression of all invertase family members. An examination of the microsyntenic regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis invertases. We also find evidence for expression of a novel intronless vacuolar invertase (PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the genome. To our knowledge, this is the first intronless invertase found in plants. The response of two poplar vacuolar invertases ( PtVIN2 and -3) to exogenous sugar treatments was examined and compared to those of the Arabidopsis, maize and rice vacuolar invertase orthologs. We found that PtVIN2 and -3 exhibit a reciprocal response to both light and exogenous sugar treatments whereby PtVIN2 expression is repressed under both light and high sugar conditions while PtVIN3 is induced under the same conditions. This reciprocal response has been previously documented in other plant systems including Arabidopsis, maize and rice. An examination of the microsyntenic chromosomal regions containing vacuolar invertase reveals extensive colinearity between Arabidopsis and poplar, but does not include rice and maize. The conserved colinear structure of the chromosomal segments containing the vacuolar invertases in Arabidopsis and poplar, coupled with the conserved reciprocal responses of these vacuolar invertases to sugar in Arabidopsis, poplar, maize and rice, has led to the hypothesis that the reciprocal nature of this sugar response arose in an ancient genome duplication event that occurred prior to the monocot and eudicot divergence on the evolutionary tree. To better understand the role of invertase in sucrose export and sink development, yeast invertase (SUC2) was ectopically expressed in a Populus tremula x P. alba hybrid. Despite the efficacy of this transgenic approach in other plant systems resulting in whole-plant shifts in carbon allocation and dramatic changes in sugar accumulation; transgenic poplars showed no whole-plant carbon allocation shifts. Metabolic analyses revealed that while most of the transgenic poplar lines did not exhibit the expected alterations in sugar accumulation, intermediates in the glyoxylate and the tricarboxylic acid cycles did fluctuate relative to the non-transgenic controls.