New methods for sorghum transformation in temperate climates.

Sorghum (Sorghum bicolor) is an emerging cereal crop in temperate climates due to its high drought tolerance and other valuable traits. Genetic transformation is an important tool for the improvement of cereals. However, sorghum is recalcitrant to genetic transformation which is almost only successful in warmer climates. Here, we test the application of two new techniques for sorghum transformation in temperate climates, namely transient transformation by Agrobacterium tumefaciens-mediated agroinfiltration and stable transformation using gold particle bombardment and leaf whorls as explants. We optimized the transient transformation method, including post-infiltration incubation of plants in the dark and using Agrobacterium grown on plates with a high cell density (OD600 = 2.0). Expression of the green fluorescence protein (GFP)-tagged endogenous sorghum gene SbDHR2 was achieved with low transformation efficiency, and our results point out a potential weakness in using this approach for localization studies. Furthermore, we succeeded in the production of callus and somatic embryos from leaf whorls, although no genetic transformation was accomplished with this method. Both methods show potential, even if they seem to be influenced by climatic conditions and therefore need further optimization to be applied routinely in temperate climates.

BRAVO self-confined expression through WOX5 in the Arabidopsis root stem-cell niche.

In animals and plants, stem-cell niches are local microenvironments that are tightly regulated to preserve their unique identity while communicating with adjacent cells that will give rise to specialized cell types. In the primary root of Arabidopsis thaliana, two transcription factors, BRAVO and WOX5, among others, are expressed in the stem-cell niche. Intriguingly, BRAVO, a repressor of quiescent center divisions, confines its own gene expression to the stem-cell niche, as evidenced in a bravo mutant background. Here, we propose through mathematical modeling that BRAVO confines its own expression domain to the stem-cell niche by attenuating a WOX5-dependent diffusible activator of BRAVO. This negative feedback drives WOX5 activity to be spatially restricted as well. The results show that WOX5 diffusion and sequestration by binding to BRAVO are sufficient to drive the experimentally observed confined BRAVO expression at the stem-cell niche. We propose that the attenuation of a diffusible activator can be a general mechanism acting at other stem-cell niches to spatially confine genetic activity to a small region while maintaining signaling within them and with the surrounding cells.

A Method for Rapid and Reliable Molecular Detection of Drought-Response Genes in Sorghum bicolor (L.) Moench Roots.

Drought is a major environmental stress that limits growth and productivity in agricultural ecosystems limiting crop yield worldwide. Breeding crops for enhanced drought tolerance is a priority to preserve food security on the increasing world population. Recent work in Arabidopsis has shown that vascular brassinosteroid receptor BRL3 (Brassinosteroid insensitive like-3) transcriptionally controls the production of osmoprotectant metabolites that confer drought resistance without penalizing growth, offering new and exciting possibilities for biotechnological improvement of drought-resistant crops. In cereals, understanding transcriptional responses to drought is an essential step for the production of gene-edited drought-resistant cereals. In this chapter, we present a method to analyze the transcriptional responses to drought in Sorghum bicolor (L.) Moench, our cereal of choice. Among the genes we tested, we found that drought marker gene SbDHN1 has a 1000-fold increase only after 1 day of drought, bringing possibilities for the development of molecular sensors for testing drought. Overall, this analysis is useful to set up conditions of high-throughput transcriptomic analysis of drought stressed plants before drought phenotype is observed.

Analysis of metabolic dynamics during drought stress in Arabidopsis plants.

Drought is a major cause of agricultural losses worldwide. Climate change will intensify drought episodes threatening agricultural sustainability. Gaining insights into drought response mechanisms is vital for crop adaptation to climate emergency. To date, only few studies report comprehensive analyses of plant metabolic adaptation to drought. Here, we present a multifactorial metabolomic study of early-mid drought stages in the model plant Arabidopsis thaliana. We sampled root and shoot tissues of plants subjected to water withholding over a six-day time course, including brassinosteroids receptor mutants previously reported to show drought tolerance phenotypes. Furthermore, we sequenced the root transcriptome at basal and after 5 days drought, allowing direct correlation between metabolic and transcriptomic changes and the multi-omics integration. Significant abiotic stress signatures were already activated at basal conditions in a vascular-specific receptor overexpression (BRL3ox). These were also rapidly mobilized under drought, revealing a systemic adaptation strategy driven from inner tissues of the plant. Overall, this dataset provides a significant asset to study drought metabolic adaptation and allows its analysis from multiple perspectives.

The BES1/BZR1-family transcription factor MpBES1 regulates cell division and differentiation in Marchantia polymorpha.

Brassinosteroids (BRs) play essential roles in growth and development in seed plants;1 disturbances in BR homeostasis lead to altered mitotic activity in meristems2,3 and organ boundaries4,5 and to changes in meristem determinacy.6 An intricate signaling cascade linking the perception of BRs at the plasma membrane to the regulation of master transcriptional regulators belonging to the BEH, for BES1 homologues, family7 has been described in great detail in model angiosperms. Homologs of these transcription factors are present in streptophyte algae and in land plant lineages where BR signaling or function is absent or has not yet been characterized. The genome of the bryophyte Marchantia polymorpha does not encode for BR receptors but includes one close ortholog of Arabidopsis thaliana BRI1-EMS-SUPPRESSOR 1 (AtBES1)8 and Arabidopsis thaliana BRASSINAZOLE-RESISTANT 1 (AtBZR1),9 MpBES1. Altered levels of MpBES1 severely compromised cell division and differentiation, resulting in stunted thalli that failed to differentiate adult tissues and reproductive organs. The transcriptome of Mpbes1 knockout plants revealed a significant overlap with homologous functions controlled by AtBES1 and AtBZR1, suggesting that members of this gene family share a subset of common targets. Indeed, MpBES1 behaved as a gain-of-function substitute of AtBES1/AtBZR1 when expressed in Arabidopsis, probably because it mediates conserved functions but evades the regulatory mechanisms that native counterparts are subject to. Our results show that this family of transcription factors plays an ancestral role in the control of cell division and differentiation in plants and that BR signaling likely co-opted this function and imposed additional regulatory checkpoints upon it.

Precise transcriptional control of cellular quiescence by BRAVO/WOX5 complex in Arabidopsis roots.

Understanding stem cell regulatory circuits is the next challenge in plant biology, as these cells are essential for tissue growth and organ regeneration in response to stress. In the Arabidopsis primary root apex, stem cell-specific transcription factors BRAVO and WOX5 co-localize in the quiescent centre (QC) cells, where they commonly repress cell division so that these cells can act as a reservoir to replenish surrounding stem cells, yet their molecular connection remains unknown. Genetic and biochemical analysis indicates that BRAVO and WOX5 form a transcription factor complex that modulates gene expression in the QC cells to preserve overall root growth and architecture. Furthermore, by using mathematical modelling we establish that BRAVO uses the WOX5/BRAVO complex to promote WOX5 activity in the stem cells. Our results unveil the importance of transcriptional regulatory circuits in plant stem cell development.

New Role for LRR-Receptor Kinase in Sensing of Reactive Oxygen Species.

Understanding how reactive oxygen species (ROS) are sensed could help engineer plants with better stress responses that are relying on the production of ROS. Here, we summarize the latest research in ROS signaling with focus on the discovery by Wu et al. of a leucine-rich repeat receptor kinase (LRR-RK) as a hydrogen peroxide (H2O2) sensor.

Auxin-Regulated Reversible Inhibition of TMK1 Signaling by MAKR2 Modulates the Dynamics of Root Gravitropism.

Plants are able to orient their growth according to gravity, which ultimately controls both shoot and root architecture.1 Gravitropism is a dynamic process whereby gravistimulation induces the asymmetric distribution of the plant hormone auxin, leading to asymmetric growth, organ bending, and subsequent reset of auxin distribution back to the original pre-gravistimulation situation.1-3 Differential auxin accumulation during the gravitropic response depends on the activity of polarly localized PIN-FORMED (PIN) auxin-efflux carriers.1-4 In particular, the timing of this dynamic response is regulated by PIN2,5,6 but the underlying molecular mechanisms are poorly understood. Here, we show that MEMBRANE ASSOCIATED KINASE REGULATOR2 (MAKR2) controls the pace of the root gravitropic response. We found that MAKR2 is required for the PIN2 asymmetry during gravitropism by acting as a negative regulator of the cell-surface signaling mediated by the receptor-like kinase TRANSMEMBRANE KINASE1 (TMK1).2,7-10 Furthermore, we show that the MAKR2 inhibitory effect on TMK1 signaling is antagonized by auxin itself, which triggers rapid MAKR2 membrane dissociation in a TMK1-dependent manner. Our findings suggest that the timing of the root gravitropic response is orchestrated by the reversible inhibition of the TMK1 signaling pathway at the cell surface.

Delving into the evolutionary origin of steroid sensing in plants.

Brassinosteroids (BRs) are steroid hormones that play crucial roles in plant growth, development and adaptation to shifting environmental conditions. Our current understanding of the origin, evolution and functional significance of BRs is influenced by a double-edged bias: most we know stems from studies on a single species and, on the flip side, dearth of information from a phylogenetically broad and significant array of land plants precludes well-grounded comparisons. Here, we provide an update on BR presence and sensing along land plant evolution. Furthermore, a comprehensive search in all major plant lineages reveals the widespread presence of BR-receptor related sequences, suggesting that steroid-related signals may have been functional early in the evolution of land plants.

The physiology of plant responses to drought.

Drought alone causes more annual loss in crop yield than all pathogens combined. To adapt to moisture gradients in soil, plants alter their physiology, modify root growth and architecture, and close stomata on their aboveground segments. These tissue-specific responses modify the flux of cellular signals, resulting in early flowering or stunted growth and, often, reduced yield. Physiological and molecular analyses of the model plant Arabidopsis thaliana have identified phytohormone signaling as key for regulating the response to drought or water insufficiency. Here we discuss how engineering hormone signaling in specific cells and cellular domains can facilitate improved plant responses to drought. We explore current knowledge and future questions central to the quest to produce high-yield, drought-resistant crops.

Emerging roles of vascular brassinosteroid receptors of the BRI1-like family.

Brassinosteroids (BRs) are essential hormones for plant growth and development that are perceived at the plasma membrane by a group of Leucine-Rich Repeat Receptor-Like Kinases (LRR-RLKs) of the BRASSINOSTEROID INSENSITIVE 1 (BRI1) family. The BRI1 receptor was first discovered by genetic screenings based on the dwarfism of BR-deficient plants. There are three BRI1 homologs, named BRI1-like 1, 2 and 3 (BRLs), yet only BRL1 and BRL3 behave as functional BR receptors. Whereas the BRI1 pathway operates in the majority of cells to promote growth, BRL receptor signaling operates under specific spatiotemporal constraints. Despite a wealth of information on the BRI1 pathway, data on specific BRL pathways and their biological relevance is just starting to emerge. Here, we systematically compare BRLs with BRI1 to identify any differences that could account for specific receptor functions. Understanding how vascular and cell-specific BRL receptors orchestrate plant development and adaptation to the environment will help shed light on membrane signaling and cell communication in plants, while opening up novel possibilities to improve stress adaptation without penalizing growth.

Brassinosteroid signaling in plant development and adaptation to stress.

Brassinosteroids (BRs) are steroid hormones that are essential for plant growth and development. These hormones control the division, elongation and differentiation of various cell types throughout the entire plant life cycle. Our current understanding of the BR signaling pathway has mostly been obtained from studies using Arabidopsis thaliana as a model. In this context, the membrane steroid receptor BRI1 (BRASSINOSTEROID INSENSITIVE 1) binds directly to the BR ligand, triggering a signal cascade in the cytoplasm that leads to the transcription of BR-responsive genes that drive cellular growth. However, recent studies of the primary root have revealed distinct BR signaling pathways in different cell types and have highlighted cell-specific roles for BR signaling in controlling adaptation to stress. In this Review, we summarize our current knowledge of the spatiotemporal control of BR action in plant growth and development, focusing on BR functions in primary root development and growth, in stem cell self-renewal and death, and in plant adaption to environmental stress.

MyROOT: a method and software for the semiautomatic measurement of primary root length in Arabidopsis seedlings.

Root analysis is essential for both academic and agricultural research. Despite the great advances in root phenotyping and imaging, calculating root length is still performed manually and involves considerable amounts of labor and time. To overcome these limitations, we developed MyROOT, a software for the semiautomatic quantification of root growth of seedlings growing directly on agar plates. Our method automatically determines the scale from the image of the plate, and subsequently measures the root length of the individual plants. To this aim, MyROOT combines a bottom-up root tracking approach with a hypocotyl detection algorithm. At the same time as providing accurate root measurements, MyROOT also significantly minimizes the user intervention required during the process. Using Arabidopsis, we tested MyROOT with seedlings from different growth stages and experimental conditions. When comparing the data obtained from this software with that of manual root measurements, we found a high correlation between both methods (R2  = 0.997). When compared with previous developed software with similar features (BRAT and EZ-Rhizo), MyROOT offered an improved accuracy for root length measurements. Therefore, MyROOT will be of great use to the plant science community by permitting high-throughput root length measurements while saving both labor and time.

Drought Resistance by Engineering Plant Tissue-Specific Responses.

Drought is the primary cause of agricultural loss globally, and represents a major threat to food security. Currently, plant biotechnology stands as one of the most promising fields when it comes to developing crops that are able to produce high yields in water-limited conditions. From studies of Arabidopsis thaliana whole plants, the main response mechanisms to drought stress have been uncovered, and multiple drought resistance genes have already been engineered into crops. So far, most plants with enhanced drought resistance have displayed reduced crop yield, meaning that there is still a need to search for novel approaches that can uncouple drought resistance from plant growth. Our laboratory has recently shown that the receptors of brassinosteroid (BR) hormones use tissue-specific pathways to mediate different developmental responses during root growth. In Arabidopsis, we found that increasing BR receptors in the vascular plant tissues confers resistance to drought without penalizing growth, opening up an exceptional opportunity to investigate the mechanisms that confer drought resistance with cellular specificity in plants. In this review, we provide an overview of the most promising phenotypical drought traits that could be improved biotechnologically to obtain drought-tolerant cereals. In addition, we discuss how current genome editing technologies could help to identify and manipulate novel genes that might grant resistance to drought stress. In the upcoming years, we expect that sustainable solutions for enhancing crop production in water-limited environments will be identified through joint efforts.

Overexpression of the vascular brassinosteroid receptor BRL3 confers drought resistance without penalizing plant growth.

Drought represents a major threat to food security. Mechanistic data describing plant responses to drought have been studied extensively and genes conferring drought resistance have been introduced into crop plants. However, plants with enhanced drought resistance usually display lower growth, highlighting the need for strategies to uncouple drought resistance from growth. Here, we show that overexpression of BRL3, a vascular-enriched member of the brassinosteroid receptor family, can confer drought stress tolerance in Arabidopsis. Whereas loss-of-function mutations in the ubiquitously expressed BRI1 receptor leads to drought resistance at the expense of growth, overexpression of BRL3 receptor confers drought tolerance without penalizing overall growth. Systematic analyses reveal that upon drought stress, increased BRL3 triggers the accumulation of osmoprotectant metabolites including proline and sugars. Transcriptomic analysis suggests that this results from differential expression of genes in the vascular tissues. Altogether, this data suggests that manipulating BRL3 expression could be used to engineer drought tolerant crops.

A Sizer model for cell differentiation in Arabidopsis thaliana root growth.

Plant roots grow due to cell division in the meristem and subsequent cell elongation and differentiation, a tightly coordinated process that ensures growth and adaptation to the changing environment. How the newly formed cells decide to stop elongating becoming fully differentiated is not yet understood. To address this question, we established a novel approach that combines the quantitative phenotypic variability of wild-type Arabidopsis roots with computational data from mathematical models. Our analyses reveal that primary root growth is consistent with a Sizer mechanism, in which cells sense their length and stop elongating when reaching a threshold value. The local expression of brassinosteroid receptors only in the meristem is sufficient to set this value. Analysis of roots insensitive to BR signaling and of roots with gibberellin biosynthesis inhibited suggests distinct roles of these hormones on cell expansion termination. Overall, our study underscores the value of using computational modeling together with quantitative data to understand root growth.

Paracrine brassinosteroid signaling at the stem cell niche controls cellular regeneration.

Stem cell regeneration is crucial for both cell turnover and tissue healing in multicellular organisms. In Arabidopsis roots, a reduced group of cells known as the quiescent center (QC) act as a cell reservoir for surrounding stem cells during both normal growth and in response to external damage. Although cells of the QC have a very low mitotic activity, plant hormones such as brassinosteroids (BRs) can promote QC divisions. Here, we used a tissue-specific strategy to investigate the spatial signaling requirements of BR-mediated QC divisions. We generated stem cell niche-specific receptor knockout lines by placing an artificial microRNA against BRI1 (BRASSINOSTEROID INSENSITIVE 1) under the control of the QC-specific promoter WOX5. Additionally, QC-specific knock-in lines for BRI1 and its downstream transcription factor BES1 (BRI1-EMS-SUPPRESOR1) were also created using the WOX5 promoter. By analyzing the roots of these lines, we show that BES1-mediated signaling cell-autonomously promotes QC divisions, that BRI1 is essential for sensing nearby inputs and triggering QC divisions and that DNA damage promotes BR-dependent paracrine signaling in the stem cell niche as a prerequisite to stem cell replenishment.

TOPLESS mediates brassinosteroid control of shoot boundaries and root meristem development in Arabidopsis thaliana.

The transcription factor BRI1-EMS-SUPRESSOR 1 (BES1) is a master regulator of brassinosteroid (BR)-regulated gene expression. BES1 together with BRASSINAZOLE-RESISTANT 1 (BZR1) drive activated or repressed expression of several genes, and have a prominent role in negative regulation of BR synthesis. Here, we report that BES1 interaction with TOPLESS (TPL), via its ERF-associated amphiphilic repression (EAR) motif, is essential for BES1-mediated control of organ boundary formation in the shoot apical meristem and the regulation of quiescent center (QC) cell division in roots. We show that TPL binds via BES1 to the promoters of the CUC3 and BRAVO targets and suppresses their expression. Ectopic expression of TPL leads to similar organ boundary defects and alterations in QC cell division rate to the bes1-d mutation, while bes1-d defects are suppressed by the dominant interfering protein encoded by tpl-1, with these effects respectively correlating with changes in CUC3 and BRAVO expression. Together, our data unveil a pivotal role of the co-repressor TPL in the shoot and root meristems, which relies on its interaction with BES1 and regulation of BES1 target gene expression.

Methods for Modeling Brassinosteroid-Mediated Signaling in Plant Development.

Mathematical modeling of biological processes is a useful tool to draw conclusions that are contained in the data, but not directly reachable, as well as to make predictions and select the most efficient follow-up experiments. Here we outline a method to model systems of a few proteins that interact transcriptionally and/or posttranscriptionally, by representing the system as Ordinary Differential Equations and to study the model dynamics and stationary states. We exemplify this method by focusing on the regulation by the brassinosteroid (BR) signaling component BRASSINOSTEROID INSENSITIVE1 ETHYL METHYL SULFONATE SUPPRESSOR1 (BES1) of BRAVO, a quiescence-regulating transcription factor expressed in the quiescent cells of Arabidopsis thaliana roots. The method to extract the stationary states and the dynamics is provided as a Mathematica code and requires basic knowledge of the Mathematica software to be executed.