Spatiotemporal Cytoskeleton Organizations Determine Morphogenesis Of Multicellular Trichomes In Tomato

As one of the most widely planted horticultural plants in the world,tomato(Solanum lycopersicum)is also a crop of economically importance.Trichomes are the specialized structure from plant epidermis,which exists on almost plants and play an important role in the plant’s resistance to biological stress and abiotic stress.Trichomes can be divided into single-cell type and multi-cell.Compared with the single-celled trichome on Arabidopsis thaliana,the trichomes on tomato are multicellular and can be divided into seven types.In this study,we used genetic screening and living cell imaging to quantitatively study the morphology of tomato trichomes and found that the both cytoskeletons are involved in morphological regulation of tomato trichomes.The result is as follows:1.The morphology and organization of tomato trichomes.In tomato trichomes,there are four types of glandular trichome(GT)and three types of non-glandular trichome(NGT).We found that the type Ⅱ and V trichomes represented two predominant types of NGT.The overall morphology of type Ⅱ and type V NGT was similar but they consisted of different number of cells.Majority of type Ⅱ trichomes included five to eight cells and type V trichomes displayed less varied cell number with three cells as the most predominant type.The length and width of cells within type Ⅱ trichome file became gradually reduced along the cell file.A similar trend was observed in type V trichomes.2.Visualization of actin organization at different developmental stages of tomato trichomes.To visualize how actin is spatially organized in different cells of tomato trichomes,we stably transformed tomato with the actin-binding marker Lifeact(p35S: Lifeact-e GFP)and performed live-cell imaging analyses.As the cell number increased,the transverse or oblique actin filaments gradually reduced and longitudinal actin filaments progressively increased upward.A similar trend was observed in type V trichomes.3.Organization and dynamics of microtubules in tomato trichomes.During the formation of unicellular trichome in Arabidopsis,microtubules are mostly involved in trichome branching.To understand how microtubules regulate trichomes in tomato,we performed live imaging of microtubule organization by visualizing stably transformed tomato expressing end-blocking protein(EB1a)driven by Ca MV35 s promoter.At one-cell stage,EB1a-e GFP showed as dots spreading at the tip of the cell.At the six-cell stage,EB1 a in trichome cell files showed an overall helical pattern.4.dt mutants distinctly affect different trichomes cells.To uncover the genetic and molecular mechanisms controlling the cell expansion of tomato trichomes,we examined EMS mutagenized lines.To identify causative mutations,genes defective in dt mutants encode WAVE-ARP2/3 homologs were identified using next generation sequencing-based bulked-segregant analysis(BSA)approach.5.The spatial configuration of actin filaments is altered in dt mutants.To visualize actin filament organization in dt mutants,we introduced p35S: Lifeact-e GFP into the dt-1 through stable transformation.Compared with WT,the orientation of actin filament in dt-1 lost the helix pattern in all trichome cells,and instead formed longitudinal or random actin bundles.6.Microtubules are essential for actin organization in tomato trichomes.To functionally dissect the roles of microtubules and actin filaments in tomato trichomes,we treated the tomato seedlings with 20 μM Oryzalin and 20 μM Latrunculin B respectively.With Oryzalin treatment,all trichome cells became radially swollen.Oryzalin treated cells with swollen shape exhibited increased bundling of actin cables and reduced fine actin filaments.