Untargeted mutagenesis of brassinosteroid receptor SbBRI1 confers drought tolerance by altering phenylpropanoid metabolism in Sorghum bicolor.

Drought is a critical issue in modern agriculture; therefore, there is a need to create crops with drought resilience. The complexity of plant responses to abiotic stresses, particularly in the field of brassinosteroid (BR) signalling, has been the subject of extensive research. In this study, we unveil compelling insights indicating that the BRASSINOSTEROID-INSENSITIVE 1 (BRI1) receptor in Arabidopsis and Sorghum plays a critical role as a negative regulator of drought responses. Introducing untargeted mutation in the sorghum BRI1 receptor (SbBRI1) effectively enhances the plant's ability to withstand osmotic and drought stress. Through DNA Affinity Purification sequencing (DAP-seq), we show that the sorghum BRI1-EMS-SUPPRESSOR 1 (SbBES1) transcription factor, a downstream player of the BR signalling, binds to a conserved G-box binding motif, and it is responsible for regulating BR homeostasis, as its Arabidopsis ortholog AtBES1. We further characterized the drought tolerance of sorghum bri1 mutants and decipher SbBES1-mediated regulation of phenylpropanoid pathway. Our findings suggest that SbBRI1 signalling serves a dual purpose: under normal conditions, it regulates lignin biosynthesis by SbBES1, but during drought conditions, BES1 becomes less active, allowing the activation of the flavonoid pathway. This adaptive shift improves the photosynthetic rate and photoprotection, reinforcing crop adaptation to drought.

Maize mutant screens: from classical methods to new CRISPR-based approaches.

Mutations play a pivotal role in shaping the trajectory and outcomes of a species evolution and domestication. Maize (Zea mays) has been a major staple crop and model for genetic research for more than 100 yr. With the arrival of site-directed mutagenesis and genome editing (GE) driven by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), maize mutational research is once again in the spotlight. If we combine the powerful physiological and genetic characteristics of maize with the already available and ever increasing toolbox of CRISPR-Cas, prospects for its future trait engineering are very promising. This review aimed to give an overview of the progression and learnings of maize screening studies analyzing forward genetics, natural variation and reverse genetics to focus on recent GE approaches. We will highlight how each strategy and resource has contributed to our understanding of maize natural and induced trait variability and how this information could be used to design the next generation of mutational screenings.

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.

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.

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.

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.

The Primary Root of Sorghum bicolor (L. Moench) as a Model System to Study Brassinosteroid Signaling in Crops.

Roots anchor plants to the soil and are essential for a successful plant growth and adaptation to the environment. Research on the primary root in the plant model system Arabidopsis thaliana has yielded important advances in the molecular and cellular understanding of root growth and development. Several studies have uncovered how the hormones brassinosteroids (BRs) control cell cycle and differentiation programs through different cell-specific signaling pathways that are key for root growth and development. Currently, an important challenge resides in the translation of the current knowledge on Arabidopsis roots into agronomically valuable species to improve the agricultural production and to meet the food security goals of the millennium. In this chapter, we characterize the primary root apex of the cereal Sorghum bicolor (L. Moench) (sorghum), analyze the physiological response of sorghum roots to BRs, and examine the phylogeny of the BRASSINOSTEROID INSENSITIVE1-like receptor family in Arabidopsis and its orthologous genes in sorghum. Overall, we support the use of sorghum as a suitable crop model species for the study of BR signaling in root growth and development. The methods presented enable any laboratory worldwide to use sorghum primary roots as a favorite organ for the study of growth and development in crops.