Heterologous transformation of Camelina sativa with high-speed chimeric myosin XI-2 promotes plant growth and leads to increased seed yield.

Camelina sativa is a Brassicaceae oilseed plant used as a biotechnology platform for biofuel and healthy vegetable oil. As Camelina is closely related to the model plant Arabidopsis, the genetic tools of Arabidopsis are considered useful when applied to Camelina. Myosin XI-2 is one of the major motive forces driving cytoplasmic streaming in Arabidopsis. In our previous study, high-speed chimeric myosin XI-2, a myosin XI-2 artificially modified by genetically exchanging the motor domain of Arabidopsis myosin XI-2 with the faster Chara myosin XI, was shown to accelerate cytoplasmic streaming and promote plant growth in Arabidopsis. Here, we heterologously transformed this high-speed Chara-Arabidopsis chimeric myosin XI-2 gene in Camelina. The transgenic plants exhibited not only enhancement of leaf development and main stem elongation but also early flowering and seed setting, indicating that the high-speed chimeric myosin XI-2 can improve plant growth in Camelina. Interestingly, total seed yield was significantly increased in the transgenic plants as the total seed number increased. Our results suggest that the high-speed myosin XI system might also be effective to improve the growth of other closely related plant species.

Characterization of ancestral myosin XI from Marchantia polymorpha by heterologous expression in Arabidopsis thaliana.

Previous studies have revealed duplications and diversification of myosin XI genes between angiosperms and bryophytes; however, the functional differentiation and conservation of myosin XI between them remain unclear. Here, we identified a single myosin XI gene from the liverwort Marchantia polymorpha (Mp). The molecular properties of Mp myosin XI are similar to those of Arabidopsis myosin XIs responsible for cytoplasmic streaming, suggesting that the motor function of myosin XI is able to generate cytoplasmic streaming. In cultured Arabidopsis cells, transiently expressed green fluorescent protein (GFP)-fused Mp myosin XI was observed as some intracellular structures moving along the F-actin. These intracellular structures were co-localized with motile endoplasmic reticulum (ER) strands, suggesting that Mp myosin XI binds to the ER and generates intracellular transport in Arabidopsis cells. The tail domain of Mp myosin XI was co-localized with that of Arabidopsis myosin XI-2 and XI-K, suggesting that all these myosin XIs bind to common cargoes. Furthermore, expression of GFP-fused Mp myosin XI rescued the defects of growth, cytoplasmic streaming and actin organization in Arabidopsis multiple myosin XI knockout mutants. The heterologous expression experiments demonstrated the cellular and physiological competence of Mp myosin XI in Arabidopsis. However, the average velocity of organelle transport in Marchantia rhizoids was 0.04 ± 0.01 µm s-1 , which is approximately one-hundredth of that in Arabidopsis cells. Taken together, our results suggest that the molecular properties of myosin XI are conserved, but myosin XI-driven intracellular transport in vivo would be differentiated from bryophytes to angiosperms.

Functional Diversity of Class XI Myosins in Arabidopsis thaliana.

Plant myosin XI acts as a motive force for cytoplasmic streaming through interacting with actin filaments within the cell. Arabidopsis thaliana (At) has 13 genes belonging to the myosin XI family. Previous reverse genetic approaches suggest that At myosin XIs are partially redundant, but are functionally diverse for their specific tasks within the plant. However, the tissue-specific expression and enzymatic properties of myosin XIs have to date been poorly understood, primarily because of the difficulty in cloning and expressing large myosin XI genes and proteins. In this study, we cloned full-length cDNAs and promoter regions for all 13 At myosin XIs and identified tissue-specific expression (using promoter-reporter assays) and motile and enzymatic activities (using in vitro assays). In general, myosins belonging to the same class have similar velocities and ATPase activities. However, the velocities and ATPase activities of the 13 At myosin XIs are significantly different and are classified broadly into three groups based on velocity (high group, medium group and low group). Interestingly, the velocity groups appear roughly correlated with the tissue-specific expression patterns. Generally, ubiquitously expressed At myosin XIs belong to the medium-velocity group, pollen-specific At myosin XIs belong to the high-velocity group and only one At myosin XI (XI-I) is classified as belonging to the low-velocity group. In this study, we demonstrated the diversity of the 13 myosin XIs in Arabidopsis at the molecular and tissue levels. Our results indicate that myosin XIs in higher plants have distinct motile and enzymatic activities adapted for their specific roles.

Actin-myosin XI: an intracellular control network in plants.

Actin is one of the three major cytoskeletal components in eukaryotic cells. Myosin XI is an actin-based motor protein in plant cells. Organelles are attached to myosin XI and translocated along the actin filaments. This dynamic actin-myosin XI system plays a major role in subcellular organelle transport and cytoplasmic streaming. Previous studies have revealed that myosin-driven transport and the actin cytoskeleton play essential roles in plant cell growth. Recent data have indicated that the actin-myosin XI cytoskeleton is essential for not only cell growth but also reproductive processes and responses to the environment. In this review, we have summarized previous reports regarding the role of the actin-myosin XI cytoskeleton in cytoplasmic streaming and plant development and recent advances in the understanding of the functions of actin-myosin XI cytoskeleton in Arabidopsis thaliana.

Measurement of enzymatic and motile activities of Arabidopsis myosins by using Arabidopsis actins.

There are two classes of myosin, XI and VIII, in higher plants. Myosin XI moves actin filaments at high speed and its enzyme activity is also very high. In contrast, myosin VIII moves actin filaments very slowly with very low enzyme activity. Because most of these enzymatic and motile activities were measured using animal skeletal muscle α-actin, but not plant actin, they would not accurately reflect the actual activities in plant cells. We thus measured enzymatic and motile activities of the motor domains of two Arabidopsis myosin XI isoforms (MYA2, XI-B), and one Arabidopsis myosin VIII isoform (ATM1), by using three Arabidopsis actin isoforms (ACT1, ACT2, and ACT7). The measured activities were different from those measured by using muscle actin. Moreover, Arabidopsis myosins showed different enzymatic and motile activities when using different Arabidopsis actin isoforms. Our results suggest that plant actin should be used for measuring enzymatic and motile activities of plant myosins and that different actin isoforms in plant cells might function as different tracks along which affinities and velocities of each myosin isoform are modulated.

Photoassimilation, assimilate translocation and plasmodesmal biogenesis in the source leaves of Arabidopsis thaliana grown under an increased atmospheric CO2 concentration.

Using 18-day-old Arabidopsis thaliana seedlings grown under increased (780 p.p.m., experimental plants) or ambient (390 p.p.m., control plants) CO2 conditions, we evaluated (14)CO2 photoassimilation in and translocation from representative source leaves. The total (14)CO2 photoassimilation amounts increased in the third leaves of the experimental plants in comparison with that found for the third leaves of the control plants, but the rates were comparable for the first leaves of the two groups. In contrast, translocation of labeled assimilates doubled in the first leaves of the experimental group, whereas translocation was, at best, passively enhanced even though photoassimilation increased in their third leaves. The transcript levels of the companion cell-specific sucrose:H(+) symporter gene SUC2 were not significantly affected in the two groups of plants, whereas those of the sucrose effluxer gene SWEET12 and the sieve element-targeted sucrose:H(+) symporter gene SUT4 were up-regulated in the experimental plants, suggesting up-regulation of SUT4-dependent apoplastic phloem loading. Compared with SUC2, SUT4 is a minor component that is expressed in companion cells but functions in sieve elements after transfer through plasmodesmata. The number of aniline blue-stained spots for plasmodesma-associated callose in the midrib wall increased in the first leaf of the experimental plants but was comparable in the third leaf between the experimental and control plants. These results suggest that A. thaliana responds to greater than normal concentrations of CO2 differentially in the first and third leaves in regards to photoassimilation, assimilate translocation and plasmodesmal biogenesis.