In plants, many signalling molecules, such as phytohormones, miRNAs, transcription factors, and small signalling peptides, drive growth and development. However, very few small signalling peptides have been shown to be necessary for lateral root development. Here, we describe the role of the peptide RALFL34 during early events in lateral root development, and demonstrate its specific importance in orchestrating formative cell divisions in the pericycle. Our results further suggest that this small signalling peptide acts on the transcriptional cascade leading to a new lateral root upstream of GATA23, an important player in lateral root formation. In addition, we describe a role for ETHYLENE RESPONSE FACTORs (ERFs) in regulating RALFL34 expression. Taken together, we put forward RALFL34 as a new, important player in lateral root initiation.
Two hemibiotrophic pathogens, Colletotrichum acutatum (Ca) and C . gloeosporioides (Cg), cause anthracnose fruit rot and anthracnose crown rot in strawberry ( Fragaria × ananassa Duchesne), respectively. Both Ca and Cg can initially infect through a brief biotrophic phase, which is associated with the production of intracellular primary hyphae that can infect host cells without causing cell death and establishing hemibiotrophic infection (HBI) or quiescent (latent infections) in leaf tissues. The Ca and Cg HBI in nurseries and subsequent distribution of asymptomatic infected transplants to fruit production fields is the major source of anthracnose epidemics in North Carolina. In the absence of complete resistance, strawberry varieties with good fruit quality showing rate-reducing resistance have frequently been used as a source of resistance to Ca and Cg. However, the molecular mechanisms underlying the rate-reducing resistance or susceptibility to Ca and Cg are still unknown. We performed comparative transcriptome analyses to examine how rate-reducing resistant genotype NCS 10-147 and susceptible genotype ‘Chandler’ respond to Ca and Cg and identify molecular events between 0 and 48 h after the pathogen-inoculated and mock-inoculated leaf tissues. Although plant response to both Ca and Cg at the same timepoint was not similar, more genes in the resistant interaction were upregulated at 24 hpi with Ca compared with those at 48 hpi. In contrast, a few genes were upregulated in the resistant interaction at 48 hpi with Cg. Resistance response to both Ca and Cg was associated with upregulation of MLP-like protein 44, LRR receptor-like serine/threonine-protein kinase, and auxin signaling pathway, whereas susceptibility was linked to modulation of the phenylpropanoid pathway. Gene regulatory network inference analysis revealed candidate transcription factors (TFs) such as GATA5 and MYB-10, and their downstream targets were upregulated in resistant interactions. Our results provide valuable insights into transcriptional changes during resistant and susceptible interactions, which can further facilitate assessing candidate genes necessary for resistance to two hemibiotrophic Colletotrichum spp. in strawberry.
Capturing cell-to-cell signals in a three-dimensional (3D) environment is key to studying cellular functions. A major challenge in the current culturing methods is the lack of accurately capturing multicellular 3D environments. In this study, we established a framework for 3D bioprinting plant cells to study cell viability, cell division, and cell identity. We established long-term cell viability for bioprinted Arabidopsis and soybean cells. To analyze the generated large image datasets, we developed a high-throughput image analysis pipeline. Furthermore, we showed the cell cycle reentry of bioprinted cells for which the timing coincides with the induction of core cell cycle genes and regeneration-related genes, ultimately leading to microcallus formation. Last, the identity of bioprinted Arabidopsis root cells expressing endodermal markers was maintained for longer periods. The framework established here paves the way for a general use of 3D bioprinting for studying cellular reprogramming and cell cycle reentry toward tissue regeneration.
Stem cells give rise to the entirety of cells within an organ. Maintaining stem cell identity and coordinately regulating stem cell divisions is crucial for proper development. In plants, mobile proteins, such as WUSCHEL-RELATED HOMEOBOX 5 (WOX5) and SHORTROOT (SHR), regulate divisions in the root stem cell niche. However, how these proteins coordinately function to establish systemic behaviour is not well understood. We propose a non-cell autonomous role for WOX5 in the cortex endodermis initial (CEI) and identify a regulator, ANGUSTIFOLIA (AN3)/GRF-INTERACTING FACTOR 1, that coordinates CEI divisions. Here, we show with a multi-scale hybrid model integrating ordinary differential equations (ODEs) and agent-based modeling that quiescent center (QC) and CEI divisions have different dynamics. Specifically, by combining continuous models to describe regulatory networks and agent-based rules, we model systemic behaviour, which led us to predict cell-type-specific expression dynamics of SHR, SCARECROW, WOX5, AN3 and CYCLIND6;1, and experimentally validate CEI cell divisions. Conclusively, our results show an interdependency between CEI and QC divisions.
ABSTRACT Elevated temperatures resulting from climate change adversely affect natural and crop ecosystems, necessitating the development of heat-tolerant crops. We established a framework to precisely identify wheat protein-phosphorylated sites associated with varying temperature sensitivities. We identified specific kinases primarily associated with a specific temperature, but our results also suggest a striking overlap between cold and heat signaling. We furthermore demonstrated that the phosphorylation state of a specific set of proteins creates a unique signature for heat stress tolerance. These findings can potentially aid in the identification of targets for breeding or genome editing to enhance sub/supra optimal temperature tolerance.
Abstract Drought stress is a major problem for agriculture worldwide, causing significant yield losses. Plants have developed highly flexible mechanisms to deal with drought, including organ‐ and developmental stage‐specific responses. In young leaves, growth is repressed as an active mechanism to save water and energy, increasing the chances of survival but decreasing yield. Despite its importance, the molecular basis for this growth inhibition is largely unknown. Here, we present a novel approach to explore early molecular mechanisms controlling Arabidopsis leaf growth inhibition following mild drought. We found that growth and transcriptome responses to drought are highly dynamic. Growth was only repressed by drought during the day, and our evidence suggests that this may be due to gating by the circadian clock. Similarly, time of day strongly affected the extent, specificity, and in certain cases even direction of drought‐induced changes in gene expression. These findings underscore the importance of taking into account diurnal patterns to understand stress responses, as only a small core of drought‐responsive genes are affected by drought at all times of the day. Finally, we leveraged our high‐resolution data to demonstrate that phenotypic and transcriptome responses can be matched to identify putative novel regulators of growth under mild drought.
Leaf growth is a complex, quantitative trait, controlled by a plethora of regulatory mechanisms. Diverse environmental stimuli inhibit leaf growth to cope with the perceived stress. In plant research, mannitol is often used to impose osmotic stress and study the underlying growth-repressing mechanisms. In growing leaf tissue of plants briefly exposed to mannitol-induced stress, a highly interconnected gene regulatory network is induced. However, early signalling and associated protein phosphorylation events that probably precede part of these transcriptional changes and that potentially act at the onset of mannitol-induced leaf size reduction are largely unknown. Here, we performed a proteome and phosphoproteome analysis on growing leaf tissue of Arabidopsis thaliana plants exposed to mild mannitol-induced stress and captured the fast (within the first half hour) events associated with this stress. Based on this in-depth data analysis, 167 and 172 differentially regulated proteins and phosphorylated sites were found. We provide these data sets as a community resource and we flag differentially phosphorylated proteins with described growth-regulatory functions, but we also illustrate potential novel regulators of shoot growth.
Stem cells divide and differentiate to form all of the specialized cell types in a multicellular organism. In the Arabidopsis root, stem cells are maintained in an undifferentiated state by a less mitotically active population of cells called the quiescent center (QC). Determining how the QC regulates the surrounding stem cell initials, or what makes the QC fundamentally different from the actively dividing initials, is important for understanding how stem cell divisions are maintained. Here we gained insight into the differences between the QC and the cortex endodermis initials (CEI) by studying the mobile transcription factor SHORTROOT (SHR) and its binding partner SCARECROW (SCR). We constructed an ordinary differential equation model of SHR and SCR in the QC and CEI which incorporated the stoichiometry of the SHR-SCR complex as well as upstream transcriptional regulation of SHR and SCR. Our model prediction, coupled with experimental validation, showed that high levels of the SHR-SCR complex are associated with more CEI division but less QC division. Furthermore, our model prediction allowed us to propose the putative upstream SHR regulators SEUSS and WUSCHEL-RELATED HOMEOBOX 5 and to experimentally validate their roles in QC and CEI division. In addition, our model established the timing of QC and CEI division and suggests that SHR repression of QC division depends on formation of the SHR homodimer. Thus, our results support that SHR-SCR protein complex stoichiometry and regulation of SHR transcription modulate the division timing of two different specialized cell types in the root stem cell niche.
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