Summary of Research for the Laboratories of Mike Hasegawa and Ra

Plant Drought and Salt Stress Tolerance Mechanisms

Research is focused on the identification of plant salt tolerance determinants. Plant genes are being isolated by direct functional selection as suppressors of salt-sensitive yeast mutants, as homologues of yeast genes involved in ion homeostasis or by molecular interaction with plant or yeast tolerance determinants. Additionally, mutant Arabidopsis populations are being generated by T-DNA activation tagging and screened for genotypes with altered stress responsiveness. The functionality of stress tolerance determinants is being confirmed by expression in transgenic plants based on sufficiency for stress tolerance or suppression of stress-sensitive mutants.

Stress Signaling through the Ca²⁺/Calmodulin-dependent Protein Phosphatase Calcineurin Mediates Salt Adaptation in Plants

Abstract: Calcineurin (CaN) is a Ca²⁺ - and calmodulin-dependent protein phosphatase (PP2B) that, in yeast, is an integral intermediate of a salt stress signal transduction pathway that effects NaCl tolerance through the regulation of Na⁺ influx and efflux. A truncated form of the catalytic subunit and the regulatory subunit of yeast CaN were co-expressed in transgenic tobacco plants to reconstitute a constitutively activated phosphatase in vivo. Several different transgenic lines, that expressed activated CaN, also exhibited substantial NaCl tolerance and this trait was linked to the genetic inheritance of the CaN transgenes. Enhanced capacity to survive NaCl shock, of plants expressing CaN, was similar when evaluation was conducted on seedlings in tissue culture raft vessels or plants in hydroponic culture that were transpiring actively. Root growth was less perturbed than shoot growth by NaCl in plants expressing CaN. Also, NaCl stress survival of control shoots was enhanced substantially when grafted onto roots of plants expressing CaN, further implicating a significant function of the phosphatase in the preservation of root integrity during salt shock. Together, these results indicate that in plants, like in yeast, a Ca²⁺ - and calmodulin-dependent CaN signal pathway regulates determinants of salt tolerance required for stress adaptation. Furthermore, modulation of this pathway by expression of an activated regulatory intermediate substantially enhanced salt tolerance.

Figure legend: Activated yeast CaN mediates NaCl tolerance of tobacco plants. Homozygous T₂ plants from lines PKY (expressing only npt-II) and A9 (expressing CNAtr and CNB1 as well as npt-II) were treated with 200 mM NaCl. After 7 days of salt-shock, plants were transferred to fresh nutrient solution without NaCl and left to recover for 12 days. Shown above are percent surviving seedlings of 36 progeny from each line (significantly different at the 95% level), and growth parameters of all plants surviving the NaCl treatment ą SE (6 and 28 plants from lines PKY and A9, respectively). Average fresh weights of PKY and A9 plants before the NaCl treatment were 1.63 ą 0.11 g and 1.76 ą 0.1 g, respectively. Illustrated below are 3 representative plants from lines A9 (top) and PKY (bottom) that survived NaCl shock and after the recovery period.

Last Update: 04/27/98