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from Ruan De Villiers:

Morning. Were these ERS genes entirely a novel discovery in this scRNA experiment, or could a bulk RNAseq experiment have been sufficient to discover them?

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from anonymous attendee:

Great talk Marina! It seemed to me the root cap has mostly ERS cells instead of the two states. You didnt focuss on these cells in your talk but if I got it right about them could it be those cells are more steady responders?

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From anonymous attendee:

great talk! what about overexpressing the ERS genes in the root? will that leads to more drought resistance?

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from Mohammad Salehin:

Hi Marina, great talk. Have you checked at the stemness of those ERS cells with any marker? Does drought stress increase the stemness of cell types?

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From Shanshan Wang:

nice talk! in your case, environmental stress is abitic stress, could this possibely be biotic stress?

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From Natanella Illouz-Eliaz:

Marina - amazing work! Could you identify/ predict what are the genes or TFs activating the ERS? Do you think that there can be mutants identified where the plants fail to activate ERS?

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Awesome talk. That was really eye opening. Seems to me that this system is tailor made for spatial transcriptomics. So do the nuclei stay fixed spatially within the cell? Presumably they replicate and move. Is there any indication how new nuclei are distributed?

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What governs nuclear replication? Is this day/night driven as in Chlamydamonas, is there some ancient mechanism? Do you see vestiges of cell wall formation that are non-functional, or do you think this represents an ancestral state?

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Seems to me that one might classify cells / organisms based on how they are temporally and spatially specialized. A single-celled (small) organism will have varied behaviors based on the integrated environmental experience, and that can shift over time. The Caulerpa organism has spatially defined specialized structures that are also dynamic. Multicellular organisms accomplish a similar thing when organizing into different cells. What do you think drove macro-spatial specificity? Do you think there's a specific evolutionary niche that encouraged this?

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From David Konkin:

Thank you both for terrific presentations! Alex: can you comment on hurdles / enablers for high throughput single cell experiments, i.e. 100+ samples

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Alex Marand: Cell state identification/ definition

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Alex Marand: Cell state identification/ definition

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Very interesting suggestions! Numbers are also infinite, but they are still studied and there are many questions we can ask and answer about them, as well as compare. So I guess you don't suggest not studying cell states but rather being more conservative in defining cell states? Challenging your valid claims, do you think there are states we could define, just more stringently? For example, within the same cell type, characterizing the finite/ infinite (not sure) states and what regulates the transitions between them could be valid? Or do you think that it would be impossible? What are your thoughts?

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From Olivier Martin:

For Elliot: thanks for showing such a unusual system, reminding one a bit of the "blob" that has become popular in schools. Polarization in plant cells plays an important role. Do you know much about what maintains polarization here, for instance for driving the oriented motion of chloroplasts?

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From Wenli Wang:

Hi Alex, are those ACRs associated with or affected by any histone modifications?

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Do you think you can use the multiome / atac data you've collected in Maize to model / predict transcriptional regulation in other species like Rice or Arabidopsis?

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From Rudiger Simon:

to Elliot: can you test for de novo organ formation and assess the potential role of informational molecules such as RNAs using regeneration experiments, following the Acetabularia paradigm from the 1930s?

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Elliot Meyerowitz: Thought provoking is an understatement! 

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Elliot Meyerowitz: Thought provoking is an understatement! 

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What determines the formation of diverse functions/ morphology in Caulerpa? A different chemical micro-environment/ niche around the nuclei? Is it regulated by the nuclei? How does the nuclei ‘know’ where it is or what it should do? If not- are these “chemical neighborhoods”? How do boundaries form in Caulerpa?

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From Marketa Pernisova:

Are all nuclei of the same character?

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From Ramiro Rodriguez:

Might this upper-lower levels of BR signalling might define in some way, for example interacting with other hormones the transition to cell expansion?

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From Ramiro Rodriguez:

Beatiful results and imaging! Considering the cell type specific signalling of BR en roots, the results you showes are in cortex or epidermal cells?

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From Herbert Ochan Alinaitwe

What could be the reason behind less or no H2O2 at down or tip portion of the plant roots during plant growth

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From Cesar Cuevas-Velazquez

Great talk, Jazz! I wonder if you think that metabolites that have striking effects like citric acid are being percibed as signalling molecules by different celltypes or is it mostly their contribution to overall metabolism (ie carbon source)

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From Ramiro Rodriguez:

Is the technology and equioment improving in terms of spatail resolution? Is it possible to combine this with those techniques used in expansion microscopy to increase inderectly spatial resolution?

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From Ramiro Rodriguez:

Con you recognise from the data cell wall components (polimers) and their modifications?

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From Anonymous Attendee:

How did you choose the concentrations of citrate etc to use?

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From Anonymous Attendee:

These are all acids, do you control pH of your growth media?

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From Patricia Schoppl:

Is the MALDI technique limited to specific types of plant tissues? Would it also work on a plant callus?

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From Suping Zhou:

Thanks for the presentation. What methods yo used to isolate the cortical and endodermis cells? any idea how much proteins/per cell? Thanks.

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From Qiuming Yao:

Hi Justin, thanks for presenting this work. Would there be enough materials in single cells to enrich for the phosphoproteome? Can we add more carrier cells for this purpose?

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From Juan-Jose Ripoll:

Hello Justin, great to see you again. One naive question. To what extend protein turnover/dynamics can impact your profiling and need to be taken ito consideration when designig your workflow/s?

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From Chirag Singhal:

Can reducing the stomata number be taken as a strategy to tackle biotic stress..?

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From Min-Yao Jhu:

It's fascinating to see the crosstalk between developmental and defense signaling. If we knock out the receptor for flg22, would the developmental signaling be able to influence the defense response?

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From Anonymous attendee:

You mentioned KC9's affinity for MPK6 decreases when MPK6 is pre-activated. Could you elaborate on the potential molecular mechanism behind this reduced affinity? Is it due to conformational changes in MPK6 upon activation, or something else?

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From Sara Pinto:

can you speculate on the nature of the small cells that are hijacked from the stomatal lineage when you apply the chemicals and how do the chemicals relate to the potential stem cell nature of those cells

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From Marketa Pernisova:

Do stomata develop the same way in cotyledons and in true leaves?

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From Mario Arteaga-Vazquez:

Is kc9 providing an adaptability response to plants challeneged with a pathogen for example Botrytis? In other words, does the cross-talk has an ecological response?

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Excellent talks. Do you think the fluid nature of cell type or identity could be due to positional information and how close to other cell type. Closer to one cell type, higher the effect?

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From Ramiro Rodriguez:

Great talk Kaisa, thanks! Is ABA responsible for stablishing the exodermis in those species were you find it without needing the ABA treatment (if there is any such species)?

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From Mst.Adia Sultana:

Is root structure involved in shoot or leaf biochemical aspects due to any kind of environmental stress?

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Could you elaborate on how the distinct transcriptional phenotypes of barrier cell types in chickpea are regulated and whether the observed differences in gene expression are conserved across other legume species?

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From Adeel Zafar:

Great talk. I have a question to Kaisa, you mentioned MYBs regulating suberin as downstream MYBs, shouldn't they be upstream MYBs since they are probably regulating the expression of suberin biosynthetic genes? I may be wrong.

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Could you elaborate on how the distinct transcriptional phenotypes of barrier cell types in chickpea are regulated and whether the observed differences in gene expression are conserved across other legume species?

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From Mst.Adia Sultana:

It was very informative session and I got some new ideas. We should be very careful about our environment. Thank you speakers.

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From Jennifer Lopez Ortiz:

Thanks Kaisa for the talk. Does ABA induce also lignin deposition in exodermis? If so, when there is an ABA-induced exodermis (based on suberin) in multiple cortical layers, is lignin deposition in same layers? And can you see the polar lignin cap?

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From Anonymous Attendee:

Would adding protein and metabolite information help with better definition of cell types?

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From Monica Garcia:

Hi Ken! In the Guillotin paper I noticed that there is a large number of Arabidopsis cells labeled endocortex that are a bit far from endodermis and cortex. I was wondering if this could reflect a challenge to define cell type even in the well-studied species Arabidopsis... what are your thoughts on this?

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From Maite Saura:

When comparing cell types between species using a subset of genes (e.g., one-to-one orthologs) have you assessed whether this subset equally preserves cell type identity/ structure of the clusters within each species? I'm wondering if the level of differences or similarities observed across the species could be influenced by gene selection rather than reflecting true biological divergence.

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From Imadud Din:

Actually, cell division occurs in meristem. After division, how do the cells develop into different types?

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From Parul Gupta:

Are cell-specific markers the same across different species?

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From Virginia Gigena:

Excelent talk! I have a question about gene modules: to what extent is it possible to build a gene module that defines a cell's identity, especially when studying how this specific cell type diverges in terms of evolution?

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From Luigi Di Costanzo:

What are the functions of the most common, highly conserved genes? Is there an accurate/current list?

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Excellent talks! According to you, what is the best way to annotate a cell-type/cluster if we can't validate it using in situ hybridization? (by anonymous)

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Do you see a correlation between cell type and epigenetic/chromatin state of the cell? (by Mohit Rajabhoj)

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With more tightly spaced timepoints, we might start to see diurnal differences in transcriptional profiles. Can this be filtered out as noise or does it cause more difficult problems with the analysis? (by anonymous)

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Would the OT model be equally applicable to non-developmental timecourses, e.g. responses to stress/treatments? (by Andrew Farmer)

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Can cell cycle regulators be used as good indicators of plant cell types like transcription factors? (by Mohit Rajabhoj)

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What do you thing about the role of metabolites in cell fate establishment?

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The mathematics is prettiest (theorems!) when the drift is conservative. Does that affect in practice the user's inference of trajectories? (by Olivier Martin)

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In an equilibrium system, is it still possible to couple cells together between time points? Is there any way to utilize pseudotime trajectories at each time point to help with this? (by Sean Robertson)

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Thanks a lot for presenting yours beautiful work. I have a question about the trajectory inference, what’s the strength of your method compared with monocle3 and RNA velocity ? (by Yiui TAN)

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Can you infer cell state transitions when you lose the intermediate cell states? From a population of terminally differentiated cells, can you infer hidden common ancestors? (by anonymous)

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Can we expect optimal/locally linear cell trajectories to have some kind of selective advantage? Are there signs of conservation or independent evolution of this? Do we also see it in abnormal development, like cancer or infection? (question by Leon Rauschning)

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Q: Nice talk! So, we need descriptors. From a PCAtlas persepctive website perspective, how can we standardize descriptors? What would be the best way to represent a cell type from a given study on a PCA website?

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Q: Really cool talk!

taking together all the millions of years in evolution: have we reached the entire set of cell-types and plant structures across time, or is there still room for innovation?

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I really like the idea of a reference tree for defining cell types in terms of development and morphology, but I think that’s only half the equation. The molecular level is very plastic and changes based on the environment, time of day, location in the plant, etc., and these are (largely) reversible. I wonder if we could think of cell type as a range of potential molecular states surrounding a developmental state (like a bubble around a position on the cell lineage tree). These potential states are constrained by development and other regulatory mechanisms (chromatin accessibility, methylation?), such that the state is limited to what is relevant to the cell’s function. This is alluded to in Domcke & Shendure 2023 (see figure), but I think this goes beyond developmental potential and is relevant at all dev. stages, including fully mature cells.

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Dennis- you mentioned that plants can respond anatomically to their growing conditions with the example of the Aerenchyma cells. I am very curious about the reversibility versus irreversibility of these changes in development. Can you think of examples where anatomy changes but it is a reversible process? 

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Dennis- you mentioned that plants can respond anatomically to their growing conditions with the example of the Aerenchyma cells. I am very curious about the reversibility versus irreversibility of these changes in development. Can you think of examples where anatomy changes but it is a reversible process? 

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I would like to point folks to the definition of meristem tissue we developed in the Plant Ontology.

https://browser.planteome.org/amigo/term/PO:0009013

We have worked hard to describe the various cell types and would love feedback from the community

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Sclerified chlorenchyma occurs as transcription factors may influence cells in contact with other cell types, or it's an adaptation. This complicates the "cell type" question as sessile organisms have adapted nearly unmeasurable diversity.

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Do you have more examples like the swamp plant that show immense phenotypical plasticity based on the environment it grows in (aerenchyma or no aerenchyma)?

(And what was the species you used as an example called again?)

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Do the cell types in two different species have the same identity? [Assuming the same morphology & shared evolutionary origin.]

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Use this space to share how you are currently thinking about plant cell types/states and how we could better study and define these in the future.