The MATH-BTB Protein TaMAB2 Accumulates in Ubiquitin-Containing Foci and Interacts With the Translation Initiation Machinery in Arabidopsis.

MATH-BTB proteins are known to act as substrate-specific adaptors of CUL3-based E3 ligases in the ubiquitin proteasome pathway. Their BTB domain binds to CUL3 scaffold proteins and the less conserved MATH domain targets a highly diverse collection of substrate proteins to promote their ubiquitination and subsequent degradation. In plants, a significant expansion of the MATH-BTB family occurred in the grasses. Here, we report analysis of TaMAB2, a MATH-BTB protein transiently expressed at the onset of embryogenesis in wheat. Due to difficulties in studying its role in zygotes and early embryos, we have overexpressed TaMAB2 in Arabidopsis to generate gain-of-function mutants and to elucidate interaction partners and substrates. Overexpression plants showed severe growth defects as well as disorganization of microtubule bundles indicating that TaMAB2 interacts with substrates in Arabidopsis. In tobacco BY-2 cells, TaMAB2 showed a microtubule and ubiquitin-associated cytoplasmic localization pattern in form of foci. Its direct interaction with CUL3 suggests functions in targeting specific substrates for ubiquitin-dependent degradation. Although direct interactions with tubulin could not be confimed, tandem affinity purification of TaMAB2 interactors point towards cytoskeletal proteins including tubulin and actin as well as the translation initiation machinery. The idenification of various subunits of eucaryotic translation initiation factors eIF3 and eIF4 as TaMAB2 interactors indicate regulation of translation initiation as a major function during onset of embryogenesis in plants.

Potential of Different Coleus blumei Tissues for Rosmarinic Acid Production.

Rosmarinic acid is one of the main active components of Coleus blumei and is known to have numerous health benefits. The pharmacological significance of rosmarinic acid and its production through in vitro culture has been the subject of numerous studies. Here, the ability of different tissues to accumulate rosmarinic acid and sustainability in production over long cultivation have been tested. Calli, tumours, normal roots and hairy roots were established routinely by application of plant growth regulators or by transformation with agrobacteria. The differences among the established tumour lines were highly heterogeneous. Hairy root lines showed the highest mean growth rate and consistency in rosmarinic acid production. Although some tumour lines produced more rosmarinic acid than the hairy root lines, over a long cultivation period their productivity was unstable and decreased. Further, the effects of plant growth regulators on growth and rosmarinic acid accumulation were tested. 2,4-Dichlorophenoxyacetic acid significantly reduced tumour growth and rosmarinic acid production. 1-Naphthaleneacetic acid strongly stimulated hairy root growth whilst abscisic acid strongly enhanced rosmarinic acid production. Hairy roots cultured in an airlift bioreactor exhibited the highest potential for mass production of rosmarinic acid.

A novel bipartite nuclear localization signal guides BPM1 protein to nucleolus suggesting its Cullin3 independent function.

BPM1 belongs to the MATH-BTB family of proteins, which act as substrate-binding adaptors for the Cullin3-based E3 ubiquitin ligase. MATH-BTB proteins associate with Cullin3 via the BTB domain and with the substrate protein via the MATH domain. Few BPM1-interacting proteins with different functions are recognized, however, specific roles of BPM1, depending on its cellular localization have not been studied so far. Here, we found a novel bipartite nuclear localization signal at the C-terminus of the BPM1 protein, responsible for its nuclear and nucleolar localization and sufficient to drive the green fluorescent protein and cytoplasmic BPM4 protein into the nucleus. Co-localization analysis in live Nicotiana tabacum BY2 cells indicates a Cullin3 independent function since BPM1 localization is predominantly nucleolar and thus devoid of Cullin3. Treatment of BY2 cells with the proteasome inhibitor MG132 blocks BPM1 and Cullin3 degradation, suggesting turnover of both proteins through the ubiquitin-proteasome pathway. Possible roles of BPM1 in relation to its in vivo localization are discussed.

Rosmarinic acid synthesis in transformed callus culture of Coleus blumei benth.

Agrobacteria mediated Coleus blumei tumour tissues were cultured in vitro on MS medium. Sixteen diversified transformed callus cultures were maintained for several years in the absence of plant growth regulators and antibiotics without affecting the growth rate. Rosmarinic acid was detected spectrophotometrically in all tissue lines but in different quantities. The highest rosmarinic acid accumulation detected was 11% of dry tissue mass. The relation between culture growth and rosmarinic acid production was investigated in three callus lines. The lines showed different rosmarinic acid accumulation in relation to their growth rate; it was either parallel or inversely related to the tissue growth. The effects of certain medium constituents on the callus growth and rosmarinic acid accumulation were examined in four tumour cell lines. Addition of 4% or 5% sucrose stimulated rosmarinic acid synthesis and decreased callus growth. Nitrogen reduction to one half or one quarter of initial concentration did not affect rosmarinic acid synthesis and decreased callus growth in three lines, while it increased rosmarinic acid accumulation and callus growth in one line. Addition of 0.1 mg/l Phe stimulated rosmarinic acid production in two lines but had little effect on the rosmarinic acid level in others. Rosmarinic acid production was significantly improved on modified macronutrients, where the Ac2 line produced 16.5 mg of rosmarinic acid per tube (0.2 g of dry wt) after being in culture for 35 days.