Fig. S1 Longitudinal sections of the primary stem of the wild type and axr2-1 stained for amyloplasts. The wild type (left) and axr2-1 (right) stem segments

LAZY1 is a protein involved in gravity signaling of shoot gravitropism of rice, maize and Arabidopsis. Although the lazy1 mutants have been well-characterized, the function of the LAZY1 protein is still largely unknown. In this study, we used fluorescence microscopy to examine the subcellular localization of Arabidopsis LAZY1 (AtLAZY1) and its truncated proteins fused to GFP in tobacco leaves. We found that AtLAZY1 localizes to the plasma membrane through the C-terminal region, suggesting that the putative trans-membrane domain in the N-terminal half is not required for localization. Next, we took a biochemical approach to investigate the membrane association of AtLAZY1. Transiently expressed AtLAZY1 in transgenic Arabidopsis was fractionated in an insoluble fraction that contained membranous compartments. AtLAZY1 was solubilized by a non-ionic detergent or at a high pH condition, suggesting that AtLAZY1 is a peripheral membrane protein. We also found that when expressed in tobacco the C-terminal part of AtLAZY1 co-localized with microtubules. A microtubule binding assay showed that the C-terminal half of AtLAZY1, which localized to the plasma membrane, interacted with microtubules in vitro. These results suggest that AtLAZY1 may function with microtubules at the periphery of the plasma membrane in the gravity signaling process.

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