C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 contributes to GA-mediated growth and flowering by interaction with DELLA proteins.

Gibberellic acid (GA) plays a central role in many plant developmental processes and is crucial for crop improvement. DELLA proteins, the core suppressors in the GA signaling pathway, are degraded by GA via the 26S proteasomal pathway to release the GA response. However, little is known about the phosphorylation-mediated regulation of DELLA proteins. In this study, we combined GA response assays with protein-protein interaction analysis to infer the connection between Arabidopsis thaliana DELLAs and the C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), a phosphatase involved in the dephosphorylation of RNA polymerase II. We show that CPL3 directly interacts with DELLA proteins and promotes DELLA protein stability by inhibiting its degradation by the 26S proteasome. Consequently, CPL3 negatively modulates multiple GA-mediated processes of plant development, including hypocotyl elongation, flowering time, and anthocyanin accumulation. Taken together, our findings demonstrate that CPL3 serves as a novel regulator that could improve DELLA stability and thereby participate in GA signaling transduction.

Single-cell transcriptomics reveal heterogeneity in plant responses to the environment: a focus on biotic and abiotic interactions.

Environmental cues, from biotic or abiotic origin, are major factors influencing plant growth and productivity. Interactions with biotic (e.g. symbionts and pathogens) and abiotic (e.g. changes in temperature, water or nutrient availability) factors trigger signaling and downstream transcriptome changes in plants. While bulk RNA-sequencing technologies have traditionally been used to profile these transcriptional changes, the heterogeneity of the responses, caused by the cellular complexity of organs, might be masked by homogenizing tissues. Thus, whether different cell types respond equally to environmental fluctuations, or whether subsets of the responses are cell-type specific, are long-lasting questions in plant biology. The recent break-through of single-cell transcriptomics in plant research offers an unprecedented view on cellular responses under changing environmental conditions. In this review, we discuss the contributions of single-cell transcriptomics towards the understanding of cell-type specific plant responses to biotic and abiotic environmental interactions. Besides major biological findings, we present some technical challenges coupled to single-cell studies of plant-environment interactions, proposing possible solutions and exciting paths for future research.

Increasing yield on dry fields: molecular pathways with growing potential.

Drought stress constitutes one of the major constraints to agriculture all over the world, and its devastating effect is only expected to increase in the following years due to climate change. Concurrently, the increasing food demand in a steadily growing population requires a proportional increase in yield and crop production. In the past, research aimed to increase plant resilience to severe drought stress. However, this often resulted in stunted growth and reduced yield under favorable conditions or moderate drought. Nowadays, drought tolerance research aims to maintain plant growth and yield under drought conditions. Overall, recently deployed strategies to engineer drought tolerance in the lab can be classified into a 'growth-centered' strategy, which focuses on keeping growth unaffected by the drought stress, and a 'drought resilience without growth penalty' strategy, in which the main aim is still to boost drought resilience, while limiting the side effects on plant growth. In this review, we put the scope on these two strategies and some molecular players that were successfully engineered to generate drought-tolerant plants: abscisic acid, brassinosteroids, cytokinins, ethylene, ROS scavenging genes, strigolactones, and aquaporins. We discuss how these pathways participate in growth and stress response regulation under drought. Finally, we present an overview of the current insights and future perspectives in the development of new strategies to improve drought tolerance in the field.

Single-cell transcriptomics sheds light on the identity and metabolism of developing leaf cells.

As the main photosynthetic instruments of vascular plants, leaves are crucial and complex plant organs. A strict organization of leaf mesophyll and epidermal cell layers orchestrates photosynthesis and gas exchange. In addition, water and nutrients for leaf growth are transported through the vascular tissue. To establish the single-cell transcriptomic landscape of these different leaf tissues, we performed high-throughput transcriptome sequencing of individual cells isolated from young leaves of Arabidopsis (Arabidopsis thaliana) seedlings grown in two different environmental conditions. The detection of approximately 19,000 different transcripts in over 1,800 high-quality leaf cells revealed 14 cell populations composing the young, differentiating leaf. Besides the cell populations comprising the core leaf tissues, we identified subpopulations with a distinct identity or metabolic activity. In addition, we proposed cell-type-specific markers for each of these populations. Finally, an intuitive web tool allows for browsing the presented dataset. Our data present insights on how the different cell populations constituting a developing leaf are connected via developmental, metabolic, or stress-related trajectories.

Distinct cellular strategies determine sensitivity to mild drought of Arabidopsis natural accessions.

The worldwide distribution of Arabidopsis (Arabidopsis thaliana) accessions imposes different types of evolutionary pressures, which contributes to various responses of these accessions to environmental stresses. Responses to drought stress have mostly been studied in the Columbia accession, which is predominantly used in plant research. However, the reactions to drought stress are complex and our understanding of the responses that contribute to maintaining plant growth during mild drought (MD) is very limited. Here, we studied the mechanisms with which natural accessions react to MD at a physiological and molecular level during early leaf development. We documented variations in MD responses among natural accessions and used transcriptome sequencing of a drought-sensitive accession, ICE163, and a drought-insensitive accession, Yeg-1, to gain insights into the mechanisms underlying this discrepancy. This revealed that ICE163 preferentially induces jasmonate- and anthocyanin-related pathways, which are beneficial in biotic stress defense, whereas Yeg-1 has a more pronounced activation of abscisic acid signaling, the classical abiotic stress response. Related physiological traits, including the content of proline, anthocyanins, and reactive oxygen species, stomatal closure, and cellular leaf parameters, were investigated and linked to the transcriptional responses. We can conclude that most of these processes constitute general drought response mechanisms that are regulated similarly in drought-insensitive and -sensitive accessions. However, the capacity to close stomata and maintain cell expansion under MD appeared to be major factors that allow to better sustain leaf growth under MD.

Cell Cycle-Dependent Regulation and Function of ARGONAUTE1 in Plants.

Regulated gene expression is key to the orchestrated progression of the cell cycle. Many genes are expressed at specific points in the cell cycle, including important cell cycle regulators, plus factors involved in signal transduction, hormonal regulation, and metabolic control. We demonstrate that post-embryonic depletion of Arabidopsis (Arabidopsis thaliana) ARGONAUTE1 (AGO1), the main effector of plant microRNAs (miRNAs), impairs cell division in the root meristem. We utilized the highly synchronizable tobacco (Nicotiana tabacum) Bright yellow 2 (BY2) cell suspension to analyze mRNA, small RNAs, and mRNA cleavage products of synchronized BY2 cells at S, G2, M, and G1 phases of the cell cycle. This revealed that in plants, only a few miRNAs show differential accumulation during the cell cycle, and miRNA-target pairs were only identified for a small proportion of the more than 13,000 differentially expressed genes during the cell cycle. However, this unique set of miRNA-target pairs could be key to attenuate the expression of several transcription factors and disease resistance genes. We also demonstrate that AGO1 binds to a set of 19-nucleotide, tRNA-derived fragments during the cell cycle progression.

The viral F-box protein P0 induces an ER-derived autophagy degradation pathway for the clearance of membrane-bound AGO1.

RNA silencing is a major antiviral defense mechanism in plants and invertebrates. Plant ARGONAUTE1 (AGO1) is pivotal in RNA silencing, and hence is a major target for counteracting viral suppressors of RNA-silencing proteins (VSRs). P0 from Turnip yellows virus (TuYV) is a VSR that was previously shown to trigger AGO1 degradation via an autophagy-like process. However, the identity of host proteins involved and the cellular site at which AGO1 and P0 interact were unknown. Here we report that P0 and AGO1 associate on the endoplasmic reticulum (ER), resulting in their loading into ER-associated vesicles that are mobilized to the vacuole in an ATG5- and ATG7-dependent manner. We further identified ATG8-Interacting proteins 1 and 2 (ATI1 and ATI2) as proteins that associate with P0 and interact with AGO1 on the ER up to the vacuole. Notably, ATI1 and ATI2 belong to an endogenous degradation pathway of ER-associated AGO1 that is significantly induced following P0 expression. Accordingly, ATI1 and ATI2 deficiency causes a significant increase in posttranscriptional gene silencing (PTGS) activity. Collectively, we identify ATI1 and ATI2 as components of an ER-associated AGO1 turnover and proper PTGS maintenance and further show how the VSR P0 manipulates this pathway.

Plant growth under suboptimal water conditions: early responses and methods to study them.

Drought stress forms a major environmental constraint during the life cycle of plants, often decreasing plant yield and in extreme cases threatening survival. The molecular and physiological responses induced by drought have been the topic of extensive research during the past decades. Because soil-based approaches to studying drought responses are often challenging due to low throughput and insufficient control of the conditions, osmotic stress assays in plates were developed to mimic drought. Addition of compounds such as polyethylene glycol, mannitol, sorbitol, or NaCl to controlled growth media has become increasingly popular since it offers the advantage of accurate control of stress level and onset. These osmotic stress assays enabled the discovery of very early stress responses, occurring within seconds or minutes following osmotic stress exposure. In this review, we construct a detailed timeline of early responses to osmotic stress, with a focus on how they initiate plant growth arrest. We further discuss the specific responses triggered by different types and severities of osmotic stress. Finally, we compare short-term plant responses under osmotic stress versus in-soil drought and discuss the advantages, disadvantages, and future of these plate-based proxies for drought.

SIAMESE-RELATED1 Is Regulated Posttranslationally and Participates in Repression of Leaf Growth under Moderate Drought.

The plant cell cycle is tightly regulated by factors that integrate endogenous cues and environmental signals to adapt plant growth to changing conditions. Under drought, cell division in young leaves is blocked by an active mechanism, reducing the evaporative surface and conserving energy resources. The molecular function of cyclin-dependent kinase-inhibitory proteins (CKIs) in regulating the cell cycle has already been well studied, but little is known about their involvement in cell cycle regulation under adverse growth conditions. In this study, we show that the transcript of the CKI gene SIAMESE-RELATED1 (SMR1) is quickly induced under moderate drought in young Arabidopsis (Arabidopsis thaliana) leaves. Functional characterization further revealed that SMR1 inhibits cell division and affects meristem activity, thereby restricting the growth of leaves and roots. Moreover, we demonstrate that SMR1 is a short-lived protein that is degraded by the 26S proteasome after being ubiquitinated by a Cullin-RING E3 ubiquitin ligase. Consequently, overexpression of a more stable variant of the SMR1 protein leads to a much stronger phenotype than overexpression of the native SMR1. Under moderate drought, both the SMR1 transcript and SMR1 protein accumulate. Despite this induction, smr1 mutants do not show overall tolerance to drought stress but do show less growth inhibition of young leaves under drought. Surprisingly, the growth-repressive hormone ethylene promotes SMR1 induction, but the classical drought hormone abscisic acid does not.

From network to phenotype: the dynamic wiring of an Arabidopsis transcriptional network induced by osmotic stress.

Plants have established different mechanisms to cope with environmental fluctuations and accordingly fine-tune their growth and development through the regulation of complex molecular networks. It is largely unknown how the network architectures change and what the key regulators in stress responses and plant growth are. Here, we investigated a complex, highly interconnected network of 20 Arabidopsis transcription factors (TFs) at the basis of leaf growth inhibition upon mild osmotic stress. We tracked the dynamic behavior of the stress-responsive TFs over time, showing the rapid induction following stress treatment, specifically in growing leaves. The connections between the TFs were uncovered using inducible overexpression lines and were validated with transient expression assays. This study resulted in the identification of a core network, composed of ERF6, ERF8, ERF9, ERF59, and ERF98, which is responsible for most transcriptional connections. The analyses highlight the biological function of this core network in environmental adaptation and its redundancy. Finally, a phenotypic analysis of loss-of-function and gain-of-function lines of the transcription factors established multiple connections between the stress-responsive network and leaf growth.

Early mannitol-triggered changes in the Arabidopsis leaf (phospho)proteome reveal growth regulators.

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.

Time of day determines Arabidopsis transcriptome and growth dynamics under mild drought.

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.