Reverse genetic and cell biological approaches to the study of developmental functions of Class XI myosin in Arabidopsis thaliana

Myosin proteins function as molecular motors that drive the ATP-dependent movement of cellular components along actin filaments. Vascular plants encode two different types of myosin, referred to as class VIII and class XI. Although class XI myosins have been suggested to function in organelle movement and cytoplasmic streaming, little is known about their cellular function in detail.

The Arabidopsis genome encodes 13 class XI myosin genes. The reasons for the relatively large number of myosin XI isoforms present within a single plant species are unknown. To investigate the function of these gene products in the cell, we determined the spatial and temporal gene expression patterns by constructing promoter-reporter plants. Myosin genes are expressed in a variety of tissues with substantial overlap between family members. To study the biological function more intensively, homozygous T-DNA insertion lines were isolated for all 13 genes. Interestingly, five mutants showed phenotypes related to root hairs. mya2, xi-b, and xi-k showed shorter root hairs than in wild type while xi-h and mya1 produced a higher density of root hairs on the epidermis. MYA1 and XI-K are the most similar isoforms among the 13 myosins and their double mutant showed an additive phenotype with extremely short root hairs suggesting that these two myosins have partially redundant functions. Interestingly organelle movements, especially those of peroxisomes, were reduced in mya1 xi-k.

Tip growth is the key growth mechanism in root hairs and pollen tubes. Many kinds of vesicles are trafficking toward (or backward from) the apical dome of root hairs to supply membrane and cell wall material as well as energy for growing tips. These movements along the shank of the hair occurred with velocities around 2 to 3 μm/s for Arabidopsis thaliana. In xi-k mutants, root hairs grew more slowly and terminated sooner than in wild type. Interestingly, this reduction of growth rate was correlated with a fluctuation of YFP-RAbA4b accumulation at the tip of growing root hairs. Other markers, including PI4P lipid and ER, as well as calcium and actin dynamics did not show significant differences. A YFP-XI-K construct driven by its native promoter could rescue the mutant phenotype and revealed accumulation of this myosin in the tip of growing root hairs. The distribution of YFP-XI-K in the root hair tip partially overlapped with CFP-RHD4-labeled vesicles at the subapex and YFP-RabA4b vesicles at the apex of root hairs, suggesting that myosin XI-K might be involved in the accumulation of unidentified vesicles in the tip of growing root hairs.

Characterization of two mutants that showed ectopic root hair growth in the epidermis, resulting in a higher density of root hairs than wild type, mya1 and xi-h, were initiated with two analyses. At first, staining pattern of promoter-reporter constructs of three key transcription factors, WER, EGL3, and GL2 were observed in mya1. Although variation in individual samples was too large to conclude, GL2 staining patterns in mya1 occasionally were unorganized. Increasing sample population and detail study is necessary. Secondly, effects of phosphate deficiency were observed with the mya1 and the xi-h in series of phosphate concentrations ranging from 1μM to 300μM. The xi-h mutant showed insensitivity on root hair production upon phosphate deficiency, suggesting a potential function of XI-H in the response to phosphate deficiency. Confirmation of these results and further study of the MYA1 and the XI-H is essential. In summary, this study established a systematic approach to investigate the biological function of class XI myosins in plant development and significantly increases our understanding of the function of XI-K myosin in root hair tip growth.