The high efficiency of grass stomatal movement is believed to be due to their unique four-celled structure, with two dumbbell-shaped guard cells (GCs) flanked by two subsidiary cells (SCs). The molecular interaction between these cells requires further investigation. To investigate genes, including those encoding aquaporins (AQPs), expressed in maize GCs and SCs, RNA sequencing was performed on microdissected GCs and SCs from epidermis harvested during day and night. A CRISPR-based tissue-specific knockout (KO) system was then used to target ZmPIPs belonging to the plasma membrane intrinsic protein1 (PIP1) subfamily in maize GCs or SCs. A general transcriptomic analysis first revealed that genes preferentially expressed in SCs compared with GCs during the day were primarily involved in lipid transport, localization, and metabolism, while genes more highly expressed in GCs were associated with photosynthesis, chloroplasts, and plastids. Interestingly, the expression of several AQP genes varied both spatially and temporally. The KO of ZmPIP1s in GCs resulted in greater stomatal opening than wild-type plants, particularly under mild water-deficit conditions. Overall, these findings reveal transcriptomic specificities between maize GCs and SCs, providing valuable insights into the regulation of rapid stomatal movements in grasses and highlight the role of ZmPIP1s in these processes.

Nitrogen fertilization remains a cornerstone of modern agriculture, yet its excessive use contributes to environmental degradation. Rapeseed (Brassica napus L.) is notably inefficient in N uptake, highlighting the importance of root traits that enhance soil exploration and nutrient acquisition. This study investigated root transcriptomic responses to nitrate availability across rapeseed genetic diversity. A panel of 40 lines was screened on vertical agar plates, revealing substantial variation in root morphology, strong heritability, and genetic control. Low nitrate supply increased the root-to-shoot biomass ratio and stimulated lateral root proliferation. Transcriptomic profiling was then conducted on three genotype pairs selected to represent distinct root system sizes. Hydroponically grown plants were exposed to two divergent nitrate levels for 24 h, and root tissues were harvested for RNA sequencing. Differential expression analysis identified over a 1000 genes significantly induced or repressed by nitrate treatment, with only 10% shared across genotypes. Gene ontology enrichment analysis revealed a central nitrate-responsive transcriptional program, accompanied by distinct molecular signatures associated with root size. Co-expression network analysis identified regulatory modules that integrate nitrate transporters with auxin signaling and energy metabolism. These modules also uncovered roles for glucosinolate biosynthesis and aquaporin-mediated water transport. This study provides a set of candidate genes and regulatory networks that represent promising targets for breeding rapeseed varieties with optimized root traits for sustainable agriculture.

Drought events can have a devastating impact on agriculture, and due to climate change, such extreme events are expected to become more frequent. Sugarcane plays a critical role in the Brazilian economy by producing sugar and bioethanol, contributing positively to the reduction of CO2 emissions. Although sugarcane is considered resilient to drought, this stress remains the primary abiotic factor reducing sugar and biomass yields. Here, we describe the role of a sugarcane gene, ScTpx2, which is induced by drought in sugarcane leaves under field conditions. When overexpressed in Arabidopsis, ScTpx2 enhanced plant survival under extreme water deficit and improved performance under mild stress conditions, which better represent field scenarios. We subsequently overexpressed the ScTpx2 gene in sugarcane plants. After 10 days of water deficit at 30% field capacity in a greenhouse, net photosynthesis in ScTpx2-overexpressing lines (ScTpx2OE) was 12-23% higher than in wild-type plants. While malondialdehyde (MDA) content, a marker of oxidative stress, increased by 129% in wild-type plants under water deficit, in ScTpx2OE plants, the increase ranged from 20% to 107%. Additionally, the vascular bundles and xylem areas were larger in ScTpx2OE compared to WT. These findings suggest that the ScTpx2 protein influences the development of the vascular system, thereby improving water transport efficiency. Our results demonstrate that overexpression of the ScTpx2 gene mitigates the effects of water deficit in sugarcane, offering promising opportunities for biotechnological applications in developing drought-tolerant commercial cultivars.

Crop improvement goals for maize (Zea mays L.) involve the targeted optimization of various organs, making it crucial to understand the developmental characteristics and gene expression patterns during organ development and in response to environmental stresses such as drought. In this study, we investigated the development of maize leaves and internodes at both macroscopic and cellular level, and identified a shared fundamental growth design with distinct timing between the two organs. By transcriptome profiling developmental zones of leaves and internodes of different ranks, and of the ear, at different growth stages under both well-watered and drought conditions, we generated a high-resolution spatiotemporal transcriptome dataset on 272 different tissues and conditions, which we make available as a searchable database. While the gene regulatory networks governing cell division and cell elongation were highly conserved across organs, precise expression regulation of particular gene families was observed across organs and within the same organ. Additionally, we highlight the expression of key genes involved in regulating leaf angle and vascular development, showing spatiotemporal regulation of differentiation parallel to growth. This comprehensive expression atlas, combined with phenotypic data, offers a deeper understanding of the similarities and differences among shoot organs and tissues during development and drought response, and provides a valuable resource for engineering organ-specific traits in maize. ### Competing Interest Statement Authors Steven Crafts-Brandner, Jonathan Vogel and Wesley Bruce were employed by BASF Corporation, USA. The authors declare that this study received funding from BASF. The funder had the following involvement in the study: collaboratively conceived the original screening and research plans.

Plant reactions to stress vary with development stage and fitness. This study assessed the relationship between light and chilling stress in Arabidopsis acclimation. By analysing the transcriptome and proteome responses of expanding leaves subjected to varying light intensity and cold, 2251 and 2064 early response genes and proteins were identified, respectively. Many of these represent as a yet unknown part of the early response to cold, illustrating a development-dependent response to stress and duality in plant adaptations. While standard light promoted photosynthetic upregulation, plastid maintenance, and increased resilience, low light triggered a unique metabolic shift, prioritizing ribosome biogenesis and lipid metabolism and attenuating the expression of genes associated with plant immunity. The comparison of early response in young leaves with that in expanded ones showed striking differences, suggesting a sacrifice of expanded leaves to support young ones. Validations of selected DEGs in mutant background confirmed a role of HSP90-1, transcription factor FLZ13, and Phospholipase A1 (PLIP) in response to cold, and the PLIP family emerged as crucial in promoting acclimation and freezing stress tolerance. The findings highlight the dynamic mechanisms that enable plants to adapt to challenging environments and pave the way for the development of genetically modified crops with enhanced freezing tolerance.

Coumarin is a bioactive molecule that impacts various plant developmental processes, including germination, root growth and shoot elongation. Despite the long-standing awareness of coumarin’s effect on plant growth, its mode of action remains elusive. We investigated the capacity of coumarin to enhance hypocotyl elongation in light-grown Arabidopsis seedlings, demonstrating that this effect is mediated by the stimulation of cell elongation rather than cell division. Functional analyses revealed that the promotion of cell elongation occurs independently of ethylene and gibberellic acid, while emphasizing the critical roles of auxin and brassinosteroids in this process. The auxin dependency was evidenced by the reduced response to coumarin treatment in both the tir1-1/afb2-1/afb3-1 triple and slr-1 gain-of-function mutant. The persistence of coumarin’s effects in the presence of yucasin suggests that de novo auxin biosynthesis is not essential for its activity. In contrast, brassinosteroid biosynthesis is critical as coumarin failed to restore the stunted hypocotyl phenotype when brassinosteroid biosynthesis was inhibited by propiconazole. Furthermore, the brassinosteroid receptor mutant bri1-116 exhibited insensitivity to coumarin and the bak1-4 signalling mutant showed a markedly diminished response, underscoring the necessity of both brassinosteroids and a functional brassinosteroid signalling pathway for coumarin-induced hypocotyl elongation.

The plasma membrane aquaporin ZmPIP1;6 is expressed in maize stomatal complexes, with higher expression during the day than at night. To elucidate the role of ZmPIP1;6 in gas exchange and stomatal movement, it was expressed in maize (inbred line B104) under the control of the p35S promoter (overexpression) or its native promoter fused with mYFP (monomeric yellow fluorescent protein) cDNA (mYFP-ZmPIP1;6). In stomatal complexes of the mature zone of the leaf, mYFP-ZmPIP1;6 showed higher expression in subsidiary cells than in guard cells, and light and dark treatments influenced its subcellular localization. Notably, ZmPIP1;6 internalization increased in dark conditions compared with light. Stomatal opening was greater in ZmPIP1;6 overexpression plants than in the wild type, while closure exhibited greater sensitivity to elevated CO2 concentration or abscisic acid (ABA) treatment. Our findings revealed that reactive oxygen species (H2O2) were involved in ABA-induced stomatal closure, while ZmPIP1;6 was unable to facilitate H2O2 diffusion when expressed in yeast. Finally, ZmPIP1;6 overexpression and mYFP-ZmPIP1;6 transgenic plants exhibited higher abaxial stomatal density than the wild type. Overall, these results indicate that ZmPIP1;6 plays important roles in stomatal opening and CO2- and ABA-induced stomatal closure.

The nuclear matrix is a nuclear compartment that has diverse functions in chromatin regulation and transcription. However, how this structure influences epigenetic modifications and gene expression in plants is largely unknown. In this study, we show that a nuclear matrix binding protein, AHL22, together with the two transcriptional repressors FRS7 and FRS12, regulates hypocotyl elongation by suppressing the expression of a group of genes known as SMALL AUXIN UP RNAs ( SAURs ) in Arabidopsis thaliana . The transcriptional repression of SAURs depends on their attachment to the nuclear matrix. The AHL22 complex not only brings these SAURs, which contain matrix attachment regions (MARs), to the nuclear matrix, but it also recruits the histone deacetylase HDA15 to the SAUR loci. This leads to the removal of H3 acetylation at the SAUR loci and the suppression of hypocotyl elongation. Taken together, our results indicate that MAR-binding proteins act as a hub for chromatin and epigenetic regulators. Moreover, we present a mechanism by which nuclear matrix attachment to chromatin regulates histone modifications, transcription, and hypocotyl elongation.

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.

ETHYLENE RESPONSE FACTOR6 (ERF6) has emerged as a central player in stress-induced plant growth inhibition. It orchestrates complex pathways that enable plants to acclimate and thrive in challenging environments. In response to various abiotic and biotic stresses, ERF6 is promptly activated through both ethylene-dependent and -independent pathways, and contributes to enhanced stress tolerance mechanisms by activating a broad spectrum of genes at various developmental stages. Despite the crucial role of ERF6, there is currently a lack of published comprehensive insights into its function in plant growth and stress response. In this respect, based on the tight connection between ethylene and ERF6, we review the latest research findings on how ethylene regulates stress responses and the mechanisms involved. In addition, we summarize the trends and advances in ERF6-mediated plant performance under optimal and stressful conditions. Finally, we also highlight key questions and suggest potential paths to unravel the ERF6 regulon in future research.

Drought is one of the most devastating causes of yield losses in crops like maize, and the anticipated increases in severity and duration of drought spells due to climate change pose an imminent threat to agricultural productivity. To understand the drought response, phenotypic and molecular studies are typically performed at a given time point after drought onset, representing a steady-state adaptation response. Because growth is a dynamic process, we monitored the drought response with high temporal resolution and examined cellular and transcriptomic changes after rehydration at 4 and 6 days after leaf four appearance. These data showed that division zone activity is a determinant for full organ growth recovery upon rehydration. Moreover, a prolonged maintenance of cell division by the ectopic expression of PLASTOCHRON1 extends the ability to resume growth after rehydration. The transcriptome analysis indicated that GROWTH-REGULATING FACTORS (GRFs) affect leaf growth by impacting cell division duration, which was confirmed by a prolonged recovery potential of the GRF1-overexpression line after rehydration. Finally, we used a multiplex genome editing approach to evaluate the most promising differentially expressed genes from the transcriptome study and as such narrowed down the gene space from 40 to seven genes for future functional characterization.

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.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Hyperspectral imaging is a promising tool for non-destructive phenotyping of plant physiological traits, which has been transferred from remote to proximal sensing applications, and from manual laboratory setups to automated plant phenotyping platforms. Due to the higher resolution in proximal sensing, illumination variation and plant geometry result in increased non-biological variation in plant spectra that may mask subtle biological differences. Here, a better understanding of spectral measurements for proximal sensing and their application to study drought, developmental and diurnal responses was acquired in a drought case study of maize grown in a greenhouse phenotyping platform with a hyperspectral imaging setup. The use of brightness classification to reduce the illumination-induced non-biological variation is demonstrated, and allowed the detection of diurnal, developmental and early drought-induced changes in maize reflectance and physiology. Diurnal changes in transpiration rate and vapor pressure deficit were significantly correlated with red and red-edge reflectance. Drought-induced changes in effective quantum yield and water potential were accurately predicted using partial least squares regression and the newly developed Water Potential Index 2, respectively. The prediction accuracy of hyperspectral indices and partial least squares regression were similar, as long as a strong relationship between the physiological trait and reflectance was present. This demonstrates that current hyperspectral processing approaches can be used in automated plant phenotyping platforms to monitor physiological traits with a high temporal resolution.

Understanding the underlying mechanisms of plant development is crucial to successfully steer or manipulate plant growth in a targeted manner. Leaves, the primary sites of photosynthesis, are vital organs for many plant species, and leaf growth is controlled by a tight temporal and spatial regulatory network. In this review, we focus on the genetic networks governing leaf cell proliferation, one major contributor to final leaf size. First, we provide an overview of six regulator families of leaf growth in Arabidopsis: DA1, PEAPODs, KLU, GRFs, the SWI/SNF complexes, and DELLAs, together with their surrounding genetic networks. Next, we discuss their evolutionary conservation to highlight similarities and differences among species, because knowledge transfer between species remains a big challenge. Finally, we focus on the increase in knowledge of the interconnectedness between these genetic pathways, the function of the cell cycle machinery as their central convergence point, and other internal and environmental cues.

BACKGROUND:Thermography is a popular tool to assess plant water-use behavior, as plant temperature is influenced by transpiration rate, and is commonly used in field experiments to detect plant water deficit. Its application in indoor automated phenotyping platforms is still limited and mainly focuses on differences in plant temperature between genotypes or treatments, instead of estimating stomatal conductance or transpiration rate. In this study, the transferability of commonly used thermography analysis protocols from the field to greenhouse phenotyping platforms was evaluated. In addition, the added value of combining thermal infrared (TIR) with hyperspectral imaging to monitor drought effects on plant transpiration rate (E) was evaluated.RESULTS:The sensitivity of commonly used TIR indices to detect drought-induced and genotypic differences in water status was investigated in eight maize inbred lines in the automated phenotyping platform PHENOVISION. Indices that normalized plant temperature for vapor pressure deficit and/or air temperature at the time of imaging were most sensitive to drought and could detect genotypic differences in the plants' water-use behavior. However, these indices were not strongly correlated to stomatal conductance and E. The canopy temperature depression index, the crop water stress index and the simplified stomatal conductance index were more suitable to monitor these traits, and were consequently used to develop empirical E prediction models by combining them with hyperspectral indices and/or environmental variables. Different modeling strategies were evaluated, including single index-based, machine learning and mechanistic models. Model comparison showed that combining multiple TIR indices in a random forest model can improve E prediction accuracy, and that the contribution of the hyperspectral data is limited when multiple indices are used. However, the empirical models trained on one genotype were not transferable to all eight inbred lines.CONCLUSION:Overall, this study demonstrates that existing TIR indices can be used to monitor drought stress and develop E prediction models in an indoor setup, as long as the indices normalize plant temperature for ambient air temperature or relative humidity.

As agricultural production is reaching its limits regarding outputs and land use, the need to further improve crop yield is greater than ever. The limited translatability from in vitro lab results into more natural growth conditions in soil remains problematic. Although considerable progress has been made in developing soil-growth assays to tackle this bottleneck, the majority of these assays use pots or whole trays, making them not only space- and resource-intensive, but also hampering the individual treatment of plants. Therefore, we developed a flexible and compact screening system in which individual seedlings are grown in wells filled with soil. The combination of an insert plate, containing the wells, with an adapter plate, containing reservoirs, allows for single-plant irrigation, different liquid treatments and mimicking stress conditions. The system makes use of an automated image-analysis pipeline that extracts multiple growth parameters from individual seedlings over the time course of the experiment, including projected rosette area, relative growth rate, compactness, and stockiness. The system is also optimized for maize with results that are consistent with Arabidopsis while different in amplitude. We conclude that the PhenoWell® system enables the translation of results obtained from in vitro studies into useful applications in soil.

To meet the increasing global demand for food, feed, fibre and other plant-derived products, a steep increase in crop productivity is a scientifically and technically challenging imperative. The CropBooster-P project, a response to the H2020 call 'Future proofing our plants', is developing a roadmap for plant research to improve crops critical for the future of European agriculture by increasing crop yield, nutritional quality, value for non-food applications and sustainability. However, if we want to efficiently improve crop production in Europe and prioritize methods for crop trait improvement in the coming years, we need to take into account future socio-economic, technological and global developments, including numerous policy and socio-economic challenges and constraints. Based on a wide range of possible global trends and key uncertainties, we developed four extreme future learning scenarios that depict complementary future developments. Here, we elaborate on how the scenarios could inform and direct future plant research, and we aim to highlight the crop improvement approaches that could be the most promising or appropriate within each of these four future world scenarios. Moreover, we discuss some key plant technology options that would need to be developed further to meet the needs of multiple future learning scenarios, such as improving methods for breeding and genetic engineering. In addition, other diverse platforms of food production may offer unrealized potential, such as underutilized terrestrial and aquatic species as alternative sources of nutrition and biomass production. We demonstrate that although several methods or traits could facilitate a more efficient crop production system in some of the scenarios, others may offer great potential in all four of the future learning scenarios. Altogether, this indicates that depending on which future we are heading toward, distinct plant research fields should be given priority if we are to meet our food, feed and non-food biomass production needs in the coming decades.

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs.