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Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP–RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

 

Cytokinesis in land plants involves the formation of a cell plate that develops into the new cell wall. Callose, a β-1,3 glucan accumulates at later stages of cell plate development presumably to stabilize this delicate membrane network during expansion. Cytokinetic callose is considered specific to multicellular plant species, as it has not been detected in unicellular algae. Here we present callose at the cytokinesis junction of the unicellular charophyte, P. margaritaceum. Callose deposition at the division plane of P. margaritaceum showed distinct, spatiotemporal patterns likely representing distinct roles of this polymer in cytokinesis. Pharmacological inhibition by Endosidin 7 resulted in cytokinesis defects, consistent with the essential role for this polymer in P. margaritaceum cell division. Cell wall deposition at the isthmus zone was also affected by the absence of callose, demonstrating the dynamic nature of new wall assembly in P. margaritaceum. The identification of candidate callose synthase genes provides molecular evidence for callose biosynthesis in P. margaritaceum. The evolutionary implications of cytokinetic callose in this unicellular Zygnematopycean alga is discussed in the context of the conquest of land by plants. 

In plant cytokinesis, de novo formation of a cell plate evolving into the new cell wall partitions the cytoplasm of the dividing cell. In our earlier chemical genomics studies, we identified and characterized the small molecule endosidin-7, that specifically inhibits callose deposition at the cell plate, arresting late-stage cytokinesis in arabidopsis. Endosidin-7 has emerged as a very valuable tool for dissecting this essential plant process. To gain insights regarding its mode of action and the effects of cytokinesis inhibition on the overall plant response, we investigated the effect of endosidin-7 through a nuclear magnetic resonance spectroscopy (NMR) metabolomics approach. In this case study, metabolomics profiles of arabidopsis leaf and root tissues were analyzed at different growth stages and endosidin-7 exposure levels. The results show leaf and root-specific metabolic profile changes and the effects of endosidin-7 treatment on these metabolomes. Statistical analyses indicated that the effect of endosidin-7 treatment was more significant than the developmental impact. The endosidin-7 induced metabolic profiles suggest compensations for cytokinesis inhibition in central metabolism pathways. This study further shows that long-term treatment of endosidin-7 profoundly changes, likely via alteration of hormonal regulation, the primary metabolism of arabidopsis seedlings. Hormonal pathway-changes are likely reflecting the plant’s responses, compensating for the arrested cell division, which in turn are leading to global metabolite modulation. The presented NMR spectral data are made available through the Metabolomics Workbench, providing a reference resource for the scientific community. 

Heteromeric acetyl‐CoA carboxylase (htACCase) catalyzes the committed step ofde novofatty acid biosynthesis in most plant plastids. Plant htACCase is comprised of four subunits: α‐ and β‐carboxyltransferase (α‐ and β‐CT), biotin carboxylase, and biotin carboxyl carrier protein. Based onin vivoabsolute quantification of htACCase subunits, α‐CT is 3‐ to 10‐fold less abundant than its partner subunit β‐CT in developing Arabidopsis seeds [Wilson and Thelen,J. Proteome Res., 2018, 17 (5)]. To test the hypothesis that low expression of α‐CT limits htACCase activity and flux through fatty acid synthesisin planta, we overexpressedPisum sativum α‐CT, either with or without its C‐terminal non‐catalytic domain, in bothArabidopsis thalianaandCamelina sativa.First‐generation Arabidopsis seed of35S::Ps α‐CT(n = 25) and35S::Ps α‐CT^Δ406‐875(n = 47) were on average 14% higher in oil content (% dry weight) than wild type co‐cultivated in a growth chamber. First‐generation camelina seed showed an average 8% increase compared to co‐cultivated wild type. Biochemical analyses confirmed the accumulation ofPsα‐CT andPsα‐CT^Δ406‐875protein and higher htACCase activity in overexpression lines during early seed development. OverexpressedPsα‐CT co‐migrated with nativeAtβ‐CT during anion exchange chromatography, indicating co‐association. By successfully increasing seed oil content upon heterologous overexpression of α‐CT, we demonstrate how absolute quantitation ofin vivoprotein complex stoichiometry can be used to guide rational metabolic engineering.

 

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants. Floral organ identity is controlled by the well-known ABC model, which has been generalized to many flowering plants. Here, we report a previously uncharacterized MYB-like gene,AGAMOUS-LIKE FLOWER(AGLF), involved in flower development in the model legumeMedicago truncatula. Loss-of-function ofAGLFresults in flowers with stamens and carpel transformed into extra whorls of petals and sepals. Compared with the loss-of-function mutant of the class C geneAGAMOUS(MtAG) inM. truncatula, the defects in floral organ identity are similar betweenaglfandmtag, but the floral indeterminacy is enhanced in theaglfmutant. Knockout ofAGLFin the mutants of the class A geneMtAP1or the class B geneMtPIleads to an addition of a loss-of-C-function phenotype, reflecting a conventional relationship ofAGLFwith the canonical A and B genes. Furthermore, we demonstrate thatAGLFactivatesMtAGin transcriptional levels in control of floral organ identity. These data shed light on the conserved and diverged molecular mechanisms that control flower development and morphology among plant species.