Reprogramming of cell identities during development frequently requires changes in the chromatin state that need to be restricted to the correct cell populations. Here we identify an auxin hormone-regulated chromatin state switch that directs reprogramming from transit amplifying to primordium founder cell fate in Arabidopsis inflorescences. Upon auxin sensing, the MONOPTEROS transcription factor recruits SWI/SNF chromatin remodeling ATPases to increase accessibility of the DNA for induction of key regulators of flower primordium initiation. In the absence of the hormonal cue, auxin sensitive Aux/IAA proteins bound to MONOPTEROS block recruitment of the SWI/SNF chromatin remodeling ATPases in addition to recruiting a co-repressor/histone deacetylase complex. This simple and elegant hormone-mediated chromatin state switch is ideally suited for iterative flower primordium initiation and orchestrates additional auxin-regulated cell fate transitions. Our findings establish a new paradigm for nuclear response to auxin. They also provide an explanation for how this small molecule can direct diverse plant responses.

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plant's response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plant's response to high salinity and dehydration. A tomato (Lycopersicon esculentum) α-class PLD, LePLDα1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDα1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDα1 and AtPLDδ were found to be activated in response to salt stress. Moreover, pldα1 and pldδ single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth.

The dynamic nature of gene regulatory networks allows cells to rapidly respond to environmental change. However, the underlying temporal connections are missed, even in kinetic studies, as transcription factor (TF) binding within at least one time point is required to identify primary targets. The TF-regulated but unbound genes are dismissed as secondary targets. Instead, we report that these genes comprise transient TF–target interactions most relevant to rapid signal transduction. We temporally perturbed a master TF (Basic Leucine Zipper 1, bZIP1) and the nitrogen (N) signal it transduces and integrated TF regulation and binding data from the same cell samples. Our enabling approach could identify primary TF targets based solely on gene regulation, in the absence of TF binding. We uncovered three classes of primary TF targets: (i) poised (TF-bound but not TF-regulated), (ii) stable (TF-bound and TF-regulated), and (iii) transient (TF-regulated but not TF-bound), the largest class. Unexpectedly, the transient bZIP1 targets are uniquely relevant to rapid N signaling in planta, enriched in dynamic N-responsive genes, and regulated by TF and N signal interactions. These transient targets include early N responders nitrate transporter 2.1 and NIN-like protein 3, bound by bZIP1 at 1–5 min, but not at later time points following TF perturbation. Moreover, promoters of these transient targets are uniquely enriched with cis-regulatory motifs coinherited with bZIP1 binding sites, suggesting a recruitment role for bZIP1. This transient mode of TF action supports a classic, but forgotten, “hit-and-run” transcription model, which enables a “catalyst TF” to activate a large set of targets within minutes of signal perturbation. Significance Cellular signals evoke rapid and broad changes in gene regulatory networks. To uncover these network dynamics, we developed an approach able to monitor primary targets of a transcription factor (TF) based solely on gene regulation, in the absence of detectable binding. This enabled us to follow the transient propagation of a nitrogen (N) nutrient signal as a direct impact of the master TF Basic Leucine Zipper 1 (bZIP1). Unexpectedly, the largest class of primary targets that exhibit transient associations with bZIP1 is uniquely relevant to the rapid and dynamic propagation of the N signal. Our ability to uncover this transient network architecture has revealed the “dark matter” of dynamic N nutrient signaling in plants that has previously eluded detection.

Summary: TheCladosporium fulvum(Cf)‐4gene of tomato confers resistance to the fungusC. fulvum, expressing the corresponding avirulence (Avr)4gene, which codes for an elicitor protein. Little is known about how such mechanisms work, but previous studies have shown that elicitor recognition activates Ca²⁺signalling and protein kinases, such as mitogen‐activated protein kinase (MAPK) and calcium‐dependent protein kinase (CDPK). Here, we provide evidence that a new signalling component, the lipid second messenger phosphatidic acid (PA), is produced within a few minutes of AVR4/Cf‐4 interaction. Using transgenic tobacco cells expressing the tomatoCf‐4‐resistance gene as a model system, phospholipid signalling pathways were studied by pre‐labelling the cells with³²P_iand assaying for the formation of lipid signals after challenge with the fungal elicitor AVR4. A dramatic rapid response was an increase in³²P‐PA, together with its metabolic product diacylglycerol pyrophosphate (DGPP). AVR4 increased the levels of PA and DGPP in aCf‐4⁺‐, time‐ and dose‐dependent manner, while the non‐matching elicitor AVR9 did not trigger any response. In general, PA signalling can be triggered by two different pathways: via phospholipase D (PLD), which generates PA directly by hydrolysing structural phospholipids like phosphatidylcholine (PC), or via PLC, which generates diacylglycerol (DAG) that is subsequently phosphorylated to PA by DAG kinase (DGK). To determine the origin of the AVR4‐induced PA formation, a PLD‐specific transphosphatidylation assay and a differential³²P‐labelling protocol were used. The results clearly demonstrated that most PA was produced via the phosphorylation of DAG. Neomycin and U73122, inhibitors of PLC activity, inhibited AVR4‐induced PA accumulation, suggesting that the increase in DGK activity was because of increased PLC activity producing DAG. Lastly, evidence is provided that PLC signalling and, in particular, PA production could play a role in triggering responses, such as the AVR4‐induced oxidative burst. For example, PLC inhibitors inhibited the oxidative burst, and when PA was added to cells, an oxidative burst was induced.

The development of gene-editing technology holds tremendous potential for accelerating crop trait improvement to help us address the need to feed a growing global population. However, the delivery and access of gene-editing tools to the host genome and subsequent recovery of successfully edited plants form significant bottlenecks in the application of new plant breeding technologies. Moreover, the methods most suited to achieve a desired outcome vary substantially, depending on species' genotype and the targeted genetic changes. Hence, it is of importance to develop and improve multiple strategies for delivery and regeneration in order to be able to approach each application from various angles. The use of transient transformation and regeneration of plant protoplasts is one such strategy that carries unique advantages and challenges. Here, we will discuss the use of protoplast regeneration in the application of new plant breeding technologies and review pertinent literature on successful protoplast regeneration.