Physiological characterization of NaCl-tolerant mutants of the fern Ceratopteris richardii

Author

Dale Lynn Vogelien

Date of Award

12-1993

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Botany

Major Professor

Leslie G. Hickok

Committee Members

Robert Augé, Donald Dougall, Otto Schwartz

Abstract

The fern Ceratopteris richardii has been used successfully as a model system to select for single gene nuclear mutations that confer tolerance to NaCl. In this dissertation project two unlinked mutations, stl1 and stl2, were selected for use in comparative studies with the wild type in an attempt to ascertain the physiological basis of tolerance associated with these mutations.

Initial studies were designed to address the kinds of stress imposed by excess NaCl, namely osmotic and ionic stress. Cross-tolerance tests comparing the growth response of stl1 gametophytes with wild type to various osmotic and ionic stress agents indicated that this mutation is associated with a specific tolerance to Na⁺. In addition, stl1 displayed similar water relations and organic and inorganic solute profiles as the wild type in response to NaCl stress. Similar studies with stl2 gametophytes indicated that tolerance conferred by the stl2 mutation is largely associated with the ability to respond to ionic stress imposed by NaCl. In addition, stl2 gametophytes displayed a high level of tolerance to Na⁺ and Mg²⁺ salts, suggesting a nonspecific tolerance for cations and anions. While similar organic solute profiles were found between stl2 and the wild type, analysis of tissue ion content revealed that stl2 was associated with selective accumulation of K⁺ and reduced accumulation of Na⁺ during NaCl stress, resulting in higher K⁺/Na⁺ ratios. Additional studies showed supplementation of Ca²⁺ to the external medium can lessen the inhibitory effect of NaCl on wild type and stl1 gametophytic growth, but had no effect on stl2 gametophytes. The positive effect of Ca²⁺ on growth during salinity stress in the wild type and stl1 genotypes was found to correlate with maintenance of higher and reduced Na⁺ levels. stl2 gametophytes were also found to be highly sensitive to the level of K⁺ in the culture medium, with reduced levels of K⁺ resulting in enhanced gametophytic growth. Thus, it appears that stl2 gametophytes suffer from toxicity under normal culture conditions. Lastly, similar lipid composition was found between genotypes in the absence and presence of NaCl stress, thus discounting the possible involvement of a lipid-based mechanism of tolerance. In accordance with the cumulative results obtained for each mutant genotype and information obtained from the literature, hypotheses regarding the physiological basis of tolerance to NaCl were proposed. The stl1 mutation was suggested to be associated with enhanced compartmentation of Na⁺ within the cell, perhaps within the vacuole. It was proposed that the stl2 mutation affects a K⁺/cation influx channel located in the plasma membrane. In wild type cells selectivity of the channel for K⁺ and K⁺ uptake is influenced by extracellular Ca²⁺ binding to the channel protein. When Ca²⁺ is bound to the protein, the channel is selective for K⁺ over other cations and K⁺ uptake is enhanced. Removal or replacement of Ca²⁺ results in the loss of channel selectivity for K⁺, and consequently the movement of other monovalent cations through the channel increases. It is proposed that the mutant (stl2) protein possesses a higher affinity for Ca²⁺. This altered Ca²⁺ binding results in enhanced K⁺ uptake in the absence of NaCl stress and maintains channel selectivity for K⁺ during salinity stress. Alternatively, stl2 may no longer be sensitive to Ca⁺.

Recommended Citation

Vogelien, Dale Lynn, "Physiological characterization of NaCl-tolerant mutants of the fern Ceratopteris richardii. " PhD diss., University of Tennessee, 1993.
https://trace.tennessee.edu/utk_graddiss/10799