In plants, it is unclear how dispersed cortical microtubules are nucleated, polarized and organized in the absence of centrosomes. In Arabidopsis thaliana cells, expression of a fusion between the microtubule-end-binding protein AtEB1a and green fluorescent protein (GFP) results in labelling of spindle poles, where minus ends gather. During interphase, AtEB1a-GFP labels the microtubule plus end as a comet, but also marks the minus end as a site from which microtubules can grow and shrink. These minus-end nucleation sites are mobile, explaining how the cortical array can redistribute during the cell cycle and supporting the idea of a flexible centrosome in plants.

A major challenge in biology is to understand how buds comprising a few cells can give rise to complex plant and animal appendages like leaves or limbs. We address this problem through a combination of time-lapse imaging, clonal analysis, and computational modeling. We arrive at a model that shows how leaf shape can arise through feedback between early patterns of oriented growth and tissue deformation. Experimental tests through partial leaf ablation support this model and allow reevaluation of previous experimental studies. Our model allows a range of observed leaf shapes to be generated and predicts observed clone patterns in different species. Thus, our experimentally validated model may underlie the development and evolution of diverse organ shapes.

An important component of cellular biochemistry is the concentration of proteins and nucleic acids in non-membranous compartments1,2. These biomolecular condensates are formed from processes including liquid-liquid phase separation (LLPS). The multivalent interactions necessary for LLPS have been studied extensively in vitro1,3. However, what regulates LLPS in vivo is still poorly understood. Here, we identify an in vivo regulator of LLPS through a genetic suppressor screen for loss of function of the Arabidopsis RNA-binding protein FCA. FCA contains prion-like domains that phase-separate in vitro, and exhibits behavior in vivo consistent with phase separation. The mutant screen identified a functional requirement for a coiled coil protein, FLL2, in FCA nuclear body formation. FCA reduces transcriptional read-through by promoting proximal polyadenylation at many sites in the Arabidopsis genome3,4. FLL2 was required to promote this proximal polyadenylation, but not binding of FCA to target RNA. Ectopic expression of FLL2 increased the size and number of FCA nuclear bodies. Crosslinking with formaldehyde captured in vivo interactions between FLL2, FCA and the polymerase and nuclease modules of the RNA 3’ end processing machinery. These 3’ RNA processing components were found to colocalize with FCA in the nuclear bodies in vivo. We conclude that FLL2 promotes liquid-liquid phase separation, important for dynamics of polyadenylation complexes at specific poly A sites. Our findings show that coiled coil proteins can promote LLPS, expanding our understanding of the principles governing the in vivo dynamics of liquid-like bodies.

Xylem tracheary elements (TEs) form hollow, sap-conducting tubes kept open by thickened ribs of secondary cell wall that provide the major structural element in wood. These ribs are enriched with cellulose and lignin, molecules that utilize more atmospheric CO(2) than any other biopolymer on Earth. The thickenings form characteristic patterns (e.g., spiral and pitted) that depend upon the bundling of underlying microtubules [1, 2]. To identify microtubule-associated proteins (MAPs) involved in patterning microtubules, we optimized an in vitro system for triggering single Arabidopsis cells to differentiate synchronously into TEs. From more than 200 microtubule-implicated proteins, AtMAP70-5 was the only MAP upregulated upon, and specific to, TE differentiation. It lines the borders of each microtubule bundle and forms C-shaped "spacers" between adjacent bundles. Manipulating levels of AtMAP70-5 and its binding partner AtMAP70-1 by overexpression or RNA interference (RNAi) silencing shifted the balance between the characteristic patterns. RNAi silencing produced stunted plants with disorganized vascular bundles. In culture, RNAi knockdown caused ribs of secondary cell wall, surrounded by microtubules, to invaginate and fall into the cytoplasm. These results suggest that AtMAP70-5 and AtMAP70-1 are essential for defining where secondary cell wall polymers are applied at the cell cortex in wood-forming cells.

Plant-cell expansion is controlled by cellulose microfibrils in the wall with microtubules providing tracks for cellulose synthesizing enzymes. Microtubules can be reoriented experimentally and are hypothesized to reorient cyclically in aerial organs, but the mechanism is unclear. Here, Arabidopsis hypocotyl microtubules were labelled with AtEB1a-GFP (Arabidopsis microtubule end-binding protein 1a) or GFP-TUA6 (Arabidopsis alpha-tubulin 6) to record long cycles of reorientation. This revealed microtubules undergoing previously unseen clockwise or counter-clockwise rotations. Existing models emphasize selective shrinkage and regrowth or the outcome of individual microtubule encounters to explain realignment. Our higher-order view emphasizes microtubule group behaviour over time. Successive microtubules move in the same direction along self-sustaining tracks. Significantly, the tracks themselves migrate, always in the direction of the individual fast-growing ends, but twentyfold slower. Spontaneous sorting of tracks into groups with common polarities generates a mosaic of domains. Domains slowly migrate around the cell in skewed paths, generating rotations whose progressive nature is interrupted when one domain is displaced by collision with another. Rotary movements could explain how the angle of cellulose microfibrils can change from layer to layer in the polylamellate cell wall.

The principles by which cortical microtubules self-organize into a global template hold important implications for cell wall patterning. Microtubules move along bundles of microtubules, and neighboring bundles tend to form mobile domains that flow in a common direction. The bundles themselves move slowly and for longer than the individual microtubules, with domains describing slow rotary patterns. Despite this tendency for colinearity, microtubules have been seen to branch off extant microtubules at ;458. To examine this paradoxical behavior, we investigated whether some microtubules may be born on and grow along extant microtubule(s). The plus-end markers Arabidopsis thaliana end binding protein 1a, AtEB1a-GFP, and Arabidopsis SPIRAL1, SPR1-GFP, allowed microtubules of known polarity to be distinguished from underlying microtubules. This showed that the majority of microtubules do branch but in a direction heavily biased toward the plus end of the mother microtubule: few grow backward, consistent with the common polarity of domains. However, we also found that a significant proportion of emergent comets do follow the axes of extant microtubules, both at sites of apparent microtubule nucleation and at cross-over points. These phenomena help explain the persistence of bundles and counterbalance the tendency to branch.

One isoform of callose synthase, Glucan Synthase-Like7 (GSL7), is tightly coexpressed with two isoforms of sucrose synthase (SUS5 and SUS6) known to be confined to phloem sieve elements in Arabidopsis (Arabidopsis thaliana). Investigation of the phenotype of gsl7 mutants of Arabidopsis revealed that the sieve plate pores of stems and roots lack the callose lining seen in wild-type plants. Callose synthesis in other tissues of the plant appears to be unaffected. Although gsl7 plants show only minor phenotypic alterations during vegetative growth, flowering stems are reduced in height and all floral parts are smaller than those of wild-type plants. Several lines of evidence suggest that the reduced growth of the inflorescence is a result of carbohydrate starvation. Levels of sucrose, hexoses, and starch are lower in the terminal bud clusters of gsl7 than in those of wild-type plants. Transcript levels of "starvation" genes expressed in response to low sugars are elevated in the terminal bud clusters of gsl7 plants, at the end of the night, and during an extended night. Pulse-chase experiments with 14 CO 2 show that transport of assimilate in the flowering stem is much slower in gsl7 mutants than in wild-type plants. We suggest that the callose lining of sieve plate pores is essential for normal phloem transport because it confers favorable flow characteristics on the pores.

Splicing of plant organellar transcripts is facilitated by members of a large protein family, the pentatricopeptide repeat proteins. We have identified a pentatricopeptide repeat protein in a genetic screen for mutants resistant to inhibition of root growth by buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis and consequently named BIR6 (BSO-insensitive roots 6). BIR6 is involved in splicing of intron 1 of the mitochondrial nad7 transcript. Loss-of-function mutations in BIR6 result in a strongly reduced accumulation of fully processed nad7 transcript. This affects assembly of Complex I and results in moderate growth retardation. In agreement with disruption of Complex I function, the genes encoding alternative NADH oxidizing enzymes are induced in the mutant, and the mutant plants are less sensitive to mannitol and salt stress. Mutation in the BIR6 gene allowed normal root growth in presence of BSO and strongly attenuated depletion of glutathione content at these conditions. The same phenotype was observed with other mutants affected in function of Complex I, thus reinforcing the importance of Complex I function for cellular redox homeostasis.