Demonstration of prominent actin filaments in the root columella

The distribution of actin filaments within the gravity-sensing columella ce lls of plant roots remains poorly understood, with studies over numerous ye ars providing inconsistent descriptions of actin organization in these cell s. This uncertainty in actin organization, and thus in actin's role in grav iperception and gravisignaling, has led us to investigate actin arrangement s in the columella cells of Zen mays L., Medicago truncatula Gaertn., Linum usitatissimum L. and Nicotiana benthamiana Domin. Actin organization was e xamined using a combination of optimized immunofluorescence techniques, and an improved fluorochrome-conjugated phalloidin labeling method reliant on 3-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS) cross-linking combined with glycerol permeabilization. Confocal microscopy of root sections label ed with anti-actin antibodies revealed patterns suggestive of actin through out the columella region. These patterns included short and fragmented acti n bundles, fluorescent rings around amyloplasts and intense fluorescence or iginating from the nucleus. Additionally, confocal microscopy of MBS-stabil ized and Alexa Fluor-phalloidin-labeled root sections revealed a previously undetected state of actin organization in the columella. Discrete actin st ructures surrounded the amyloplasts and prominent actin cables radiated fro m the nuclear surface toward the cell periphery. Furthermore, the cortex of the columella cells contained fine actin bundles (or single filaments) tha t had a predominant transverse orientation. We also used confocal microscop y of plant roots expressing endoplasmic reticulum (ER)-targeted green fluor escent protein to demonstrate rapid ER movements within the columella cells , suggesting that the imaged actin network is functional. The successful id entification of discrete actin structures in the root columella cells forms the basis for advancing studies on the role of actin in gravity perception and signaling.