Masters Theses

Rana Ferrebee

Date of Award

8-2001

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Daniel M. Roberts

Committee Members

Elizabeth Howell, Albrecht von Arnim

Abstract

The transport of water across lipid bilayers is regulated in part by aquaporins. Aquaporins are integral membrane proteins that belong to the ancient major intrinsic protein (MIP) superfamily. From sequence analysis and biochemical localization studies plant aquaporins can be divided into three groups. PIPs (plasma membrane intrinsic proteins), TIPs (tonoplast intrinsic proteins) and nodulin 26-like proteins (NLMs). Nodulin 26 is a major protein of the symbiosome membrane of nitrogen-fixing nodules of soybeans where it functions as an aquaglyceroporin, mediating the transport of water as well as uncharged solutes. The biological function of nodulin 26 in the symbiosome membrane remains elusive, but a role in osmoregulation as well as metabolite transport has been proposed. Until recently, it was thought that nodulin 26 was a unique MIP with a dedicated function on the symbiosome membrane. However, it has recently become clear that other "nodulin 26-like" MIPs (NLMs) are present, not only in other legumes, but also in nonlegumonous plants and in tissues besides nodules. With the completion of the sequencing of the Arabidopsis genome we now know that there are 10 NLMs in Arabidopsis ranging from 32.1% to 62.4% identity to nodulin 26. In addition, sequence analysis suggests that many of these proteins will be aquaglyceroporins and also be targets for calcium dependent protein kinase (CDPK). In the present study we have investigated; 1) The tissue specific expression patterns of three NLMs, At-NLMl, At- NLM2, and At-NLM3 in Arabidopsis thaliana; 2) Effects of various environmental stimuli on the expression patterns of these NLMs; 3) The functions of one At-NLM3 by using transferred (T-DNA) mutagenesis technology. To help elucidate the specific function of NLMs in A. thaliana their tissue distribution patterns were evaluated. The results of RT-PCR show that At-NLMl is specifically expressed in two organs, the flowers and the roots. The expression in the roots is slightly higher than that in the flower. In contrast, At-NLM3 was found to be expressed in all organs tested, but the expression levels between tissues were different. At-NLM3 was expressed at the highest levels in roots and the stems and was expressed at lower levels in the flowers and leaves, and was nearly undetectable in the siliques. Lastly, At-NLM2 was expressed nearly evenly in all organs tested but at higher levels than At-NLMl and At-NLM3. To determine the potential involvement of At-NLMs in stress biology the analysis of At-NLMl and At-NLM3 transcript levels upon challenge with various environmental signals was performed. At-NLM3 levels showed a differential response to the applied stresses. At-NLM3 levels showed little sensitivity to mannitol, and showed a slight decrease with cold temperature and NaCl. However, At-NLM3 showed a higher sensitivity to drought, with a 2.5 fold reduction in At-NLM3 levels observed over 2.5 hours. In addition, the transcript level of At-NLM3 showed a 2.5 fold decrease with the application of the stress hormone abscissic acid (ABA). Lastly, At-NLM3 shows a substantial up regulation of 7-fold upon growth in darkness. Similar to At-NLM3, the application of 0.1 mM ABA decreased the amount of At-NLMl mRNA by four fold. However, in contrast to At-NLM3, transcript levels of At-NLMl exhibited a higher sensitivity to NaCl and decreased temperature, showing a 2- fold reduction for both treatments. The transcript levels of At-NLMl respond rapidly to NaCl treatment, showing a decrease by 30 minutes. The transcript levels began to decrease within a 30 minutes and eventually reach a four-fold reduction at the 34 hour time point. The levels of At-NLMl mRNA did not seem to be greatly affected by drought or mannitol. In addition, At-NLMl expression showed diurnal variation in expression with highest transcript levels during the light period of the photocycle and lowest levels within the dark part of the photocycle. The regulation of AtNLMl and At-NLM3 by stress/environmental stimuli suggest they play a role in adaptation to changing osmotic conditions and also are developmentally controlled by light stimuli. However, the role of these putative channel proteins in stress biology and membrane function remains unclear. To gain insight into its role in stress physiology, a T-DNA knockout mutant of At-NLM3 has been generated. Preliminary phenotype analyses were done on A. thaliana plants homozygous for the At- NLM3 knockout. Two reproducible growth defects were observed, one with green plants and one observed with etiolated plants. Preliminary analysis of the knockout mutant suggests some developmental defects including reduced stem and leaf growth, reduced stem thickness and stability, increase in the timing of bolting and developemental defects in etiolated paints. Overall, the data show that the NLMs in Arabidopsis are differentially expressed and are regulated at the transcript level by osmotic and stress stimuli.

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