75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

417 Calcium Entry Mediated by AtGLR3.3, a Plant Ionotropic Glutamate Receptor with a Broad
Agonist Profile
Zhi Qi, Nicholas Stephens, Edgar Spalding
University of Wisconsin-Madison
Rapid, transient changes in cytosolic calcium couple endogenous and external signals to appropriate cellular response
pathways in all manner of organisms. The molecular mechanisms that admit calcium across the plasma membrane of
plants during signaling processes has remained elusive. Arabidopsis glutamate receptor-like (GLR) genes, homologous
to ligand-gated ion channels of animal neuronal synapses, have been proposed as candidate genes for calcium entry
into the cell, though direct experimental evidence for such a role has been lacking. Application of low (micromolar)
concentrations of glutamate induces a large membrane depolarization and a concurrent, large rise in cytosolic calcium in
wild-type root cells. Both responses depended on external calcium concentration, consistent with both being caused by
calcium entering across the plasma membrane in response to glutamate treatment. Here we show that T-DNA insertions
in the GLR3.3 gene (two independent alleles) completely block the depolarization and calcium transient induced by
glutamate. An array of compounds was tested for GLR-agonist activity. The GLR3.3 ligand profile was found to include
six structurally disparate amino acids (glu, gly, ala, cys, ser, and asn) as well as the tripeptide glutathione. Taken together,
these results indicate a necessary role for this neuronal signaling homolog in amino acid recognition and calcium entry
into the plant cell.
418 SOB3 and ESC are Two Plant-Specific Genes Involved in Seedling Development
Ian Street, Purvi Shah, Allison Smith, Michael Neff
Washington University, St. Louis, MO, USA
SOB3 was cloned from an activation tagging screen for suppressors of the long hypocotyl phenotype of a weak
phyB allele, phyB-4. SOB3 encodes a single AT-hook containing protein with a second domain of unknown function and
is part of a large and conserved plant specific gene family. sob3-D (suppressor of phyB-4, Dominant) overexpressing
seedlings have shorter hypocotyls and as adults, develop significantly larger, rounder leaves, larger flowers and thicker
stems than the wild type. The differences in organ size are due to cell proliferation as epidermal cell size is similar to the
wild type. In addition, sob3-D plants have delayed senescence, living twice as long as the wild type. SOB3 is closely
related at the DNA level to one gene in this family, ESCAROLLA, identified in a different activation tagging screen.
esc-D plants are also suppressors of the phyB-4 hypocotyl phenotype and are similar as adults to sob3-D, suggesting
that these genes are redundant with respect to over-expression. A loss-of-function SOB3 allele (sob3-4) was generated
through an EMS intragenic suppressor screen of sob3-DphyB-4 plants. This allele induces a nonsense mutation in the
transcript before either of the conserved domains. Two other alleles, both lesions in the AT-hook DNA binding domain
were identified in this screen. These missense alleles suggest that the AT-hook DNA binding domain is important for
SOB3 function. A similar nonsense allele of ESC (esc-8) was obtained from the Seattle TILLING Project. The sob3-4
esc-8 double mutant confers a long hypocotyl in multiple fluence rates of white and blue light. This double mutant and
the esc-8 single mutant confer a long hypocotyl in far-red light when compared to the wild type or the sob3-4 single
mutant. No significant differences were observed under multiple fluence rates of red light. In plants transformed with
native promoter GUS translational fusion constructs of SOB3 or ESC, staining was observed in the seedling vasculature
and in white light grown seedlings, throughout the hypocotyl. Dark grown seedling were stained mostly in the vasculature
alone. In far-red light, GUS expression was observed predominantly in the hypocotyl. This suggests that SOB3 and ESC
protein expression varies depending on the light condition. Taken together, these data suggest SOB3 and ESC can act
redundantly to modulate hypocotyl growth inhibition in response to light.