Protein ubiquitylation is a post-translational modification (PTM) process that covalently modifies a protein substrate with either mono-ubiquitin moieties or poly-ubiquitin chains often at the lysine residues. In Arabidopsis, bioinformatic predictions have suggested that over 5% of its proteome constitutes the protein ubiquitylation system. Despite advancements in functional genomic studies in plants, only a small fraction of this bioinformatically predicted system has been functionally characterized. To expand our understanding about the regulatory function of protein ubiquitylation to that rivaling several other major systems, such as transcription regulation and epigenetics, I described the status, issues, and new approaches of protein ubiquitylation studies in plant biology. I summarized the methods utilized in defining the ubiquitylation machinery by bioinformatics, identifying ubiquitylation substrates by proteomics, and characterizing the ubiquitin E3 ligase-substrate pathways by functional genomics. Based on the functional and evolutionary analyses of the F-box gene superfamily, I proposed a deleterious duplication model for the large expansion of this family in plant genomes. Given this model, I presented new perspectives of future functional genomic studies on the plant ubiquitylation system to focus on core and active groups of Ub E3 ligase genes.

Root system architecture (RSA) influences the acquisition of heterogeneously dispersed soil nutrients. Cytokinin and C-TERMINALLY ENCODED PEPTIDE (CEP) hormones affect RSA, in part by controlling the angle of lateral root (LR) growth. Both hormone pathways converge on CEP DOWNSTREAM 1 (CEPD1) and CEPD2 to control primary root growth, however, a role for CEPDs in controlling the growth angle of LRs is unknown. Using phenotyping combined with genetic and grafting approaches, we showed that CEP hormone-mediated shallower LR growth required cytokinin synthesis and cytokinin perception in roots via ARABIDOPSIS HISTIDINE KINASE 2 (AHK2) and AHK3. Consistently, cytokinin synthesis and ahk2,3 mutants phenocopied the steeper-root phenotype of cep receptor 1 (cepr1) mutants on agar plates, and CEPR1 was required for trans-Zeatin (tZ)-type cytokinin-mediated shallower LR growth. In addition, cepd1,2 was less sensitive to CEP and tZ and showed basally steeper LRs on agar plates. Cytokinin and CEP pathway mutants were grown in rhizoboxes to define the role of these pathways in controlling RSA. Only cytokinin receptor mutants and cepd1,2 partially phenocopied the steeper-rooted phenotype of cepr1 mutants. These results show CEP and cytokinin signaling intersect to promote shallower LR growth, but additional components contribute to the cepr1 phenotype in soil.

The regulation of water loss and the spread of xylem embolism have mostly been considered separately. The development of an integrated approach taking into account the temporal dynamics and relative contributions of these mechanisms to plant drought responses is urgently needed. Do conifer species native to mesic and xeric environments display different hydraulic strategies and temporal sequences under drought? A dry-down experiment was performed on seedlings of four conifer species differing in embolism resistance, from drought-sensitive to extremely drought-resistant species. A set of traits related to drought survival was measured, including turgor loss point, stomatal closure, minimum leaf conductance and xylem embolism resistance. All species reached full stomatal closure before the onset of embolism, with all but the most drought-sensitive species presenting large stomatal safety margins, demonstrating that highly drought-resistant species do not keep their stomata open under drought conditions. Plant dry-down time to death was significantly influenced by the xylem embolism threshold, stomatal safety margin, and minimum leaf conductance, and was best explained by the newly introduced stomatal margin retention index (SMRIΨ50) which reflects the time required to cross the stomatal safety margin. The SMRIΨ50 may become a key tool for the characterization of interspecific drought survival variability in trees.

Gene co-expression networks (GCNs) have not been extensively studied in non-model plants. However, the rapid accumulation of transcriptome datasets in certain species represents an opportunity to explore underutilized network aggregation approaches. In fact, aggregated GCNs (aggGCNs) highlight robust co-expression interactions and improve functional connectivity. We applied and evaluated two different aggregation methods on public grapevine RNA-Seq datasets belonging to three different tissue conditions (leaf, berry and 'all organs'). Our results show that co-occurrence-based aggregation generally yielded the best-performing networks. We applied aggGCNs to study several TF gene families, showing their capacity of detecting both already-described and novel regulatory relationships between R2R3-MYBs, bHLH/MYC and multiple specialized metabolic pathways. Specifically, TF gene- and pathway-centered network analyses successfully ascertained the previously established role of VviMYBPA1 in controlling the accumulation of proanthocyanidins while providing insights into its novel role as a regulator of p-coumaroyl-CoA biosynthesis as well as the shikimate and aromatic amino-acid pathways. This network was validated using DNA Affinity Purification Sequencing data, demonstrating that co-expression networks of transcriptional activators can serve as a proxy of gene regulatory networks. This study presents an open repository to reproduce networks in other crops and a GCN application within the Vitviz platform, a user-friendly tool for exploring co-expression relationships.

Deterioration and impoverishment of soil, caused by environmental pollution and climate change result in reduced crop productivity. To adapt to hostile soils, plants have developed a complex network of factors involved in stress sensing, signal transduction and adaptive responses. The chemical properties of reactive oxygen and nitrogen species (ROS, RNS) allow them to participate in integrating the perception of external signals by fine-tuning protein redox regulation and signal transduction, triggering specific gene expression. In this review, we update and summarise the progress in understanding the mechanistic basis of ROS and RNS production at subcellular level in plants and their role in the regulation of ion channels/transporters, at both transcriptional level and post-translational level. We have also carried out an in silico analyses of different redox-dependent modifications of ion channels/transporters and identified Cys and Tyr targets of NO of metal transporters. Further we summarized possible ROS- and RNS-dependent sensors involved in metal stress sensing, such as kinases and phosphatases, as well as some transcription factors regulated by ROS and RNS that could be involved in metal homeostasis. Understanding the ROS and RNS-dependent signalling events is crucial to design new strategies to fortified crops, improve plant tolerance against nutritional imbalance and metal toxicity.

Premature senescence is an important factor affecting wheat yield and quality. Wheat yield can be increased by delaying senescence and prolonging the effective photosynthetic time. Previously, we found that the cis-Zeatin-O-glucosyltransferase (cZOGT1) gene plays an important role in the stay-green wheat phenotype. In this study, cZOGT1-overexpressing lines exhibited a delayed senescence phenotype, despite a significant reduction in the total cytokinin content. Further, we found that cZOGT1 interacted with TaZIP, and that a high level of cZOGT1 expression led to the suppression of TaZIP expression, which in turn, reduced abscisic acid (ABA) content. The synergistic reduction in cytokinins and ABA levels eventually caused the stay-green phenotype in cZOGT1-OE lines. This study provides a new theoretical basis to explain the mechanism underlying the wheat stay-green phenotype and provides an excellent genetic resource for wheat molecular-design breeding.

Physaria fendleri is a Brassicaceae that produces hydroxy fatty acids in its embryos; a type of oil that is very valuable and widely used in the industry of cosmetics, lubricants, biofuels, etc. Free of toxins and rich in hydroxy fatty acids, Physaria is a promising alternative to imported castor oil and is on the verge of being commercialized. This study aims to identify important biochemical step(s) for oil synthesis in Physaria, which may serve as target(s) for future crop improvement. To advance towards this goal, the endosperm composition was analyzed by LC-MS/MS to develop and validate culture conditions that mimic the development of the embryos in planta. Using developing Physaria embryos in culture and 13C-labeling, our studies revealed that: i) Physaria embryos metabolize carbon into biomass with an efficiency significantly lower than other photosynthetic embryos; ii) the plastidic malic enzyme provides 42% of the pyruvate used for de novo fatty acid synthesis, which is the highest measured so far in developing "green" oilseed embryos; and iii) Physaria uses non-conventional pathways to channel carbon into oil, namely the RuBisCO shunt which fixes CO2 released in the plastid, and the reversibility of the isocitrate dehydrogenase which provides additional carbon for fatty acid elongation.

Plants often face simultaneous abiotic and biotic stress conditions. However, physiological and transcriptional responses of plants under combined stress situations still need to be understood. Spring barley is susceptible to Fusarium Head Blight (FHB), which is strongly affected by weather conditions. We therefore studied the potential influence of drought on FHB severity and responses in three differently susceptible spring barley varieties. We found strongly reduced FHB severity in susceptible varieties under drought. The number of differentially expressed genes (DEGs) and strength of transcriptomic regulation reflected the concentration of physiological stress markers such as abscisic acid or fungal DNA contents. Infection-related gene expression is associated with susceptibility rather than resistance. Weighted gene correlation network analysis uncovered 18 modules of co-expressed genes, which reflect the pathogen or drought response in the used varieties. A generally infection-related module contained co-expressed genes for defence, programmed cell death and mycotoxin-detoxification indicating that diverse genotypes use a similar defence strategy towards FHB, albeit with different success. Further, DEGs showed co-expression in drought or genotype-associated modules correlating with measured phytohormones or the osmolyte proline. The combination of drought stress with infection led to the highest numbers of DEGs and provoked a modular composition of single stress responses rather than a specific transcriptional readout.

The vasculature along conifer needles is fundamentally different from that in angiosperm leaves containing a unique transfusion tissue inside the bundle sheath. To identify the pathway of photoassimilates from mesophyll to phloem, and the opposing pathway of nutrients from xylem to mesophyll, we used specific tracers. For symplasmic transport we applied esculin to the tip of attached pine needles and followed tracer movement down the phloem. For apoplasmic transport we let detached needles take up a membrane-impermeable contrast agent and used micro-X-ray computed tomography to map critical water exchange interfaces and domain borders. Microscopy and segmentation of X-ray data enabled us to render and quantify the functional 3D structure of the water-filled apoplasm and the complementary symplasmic domain. The transfusion tracheid system formed a sponge-like apoplasmic domain that was blocked at the bundle sheath. Transfusion parenchyma cell chains bridged this domain as tortuous symplasmic pathways with strong local anisotropy which, as the accumulation of esculin, pointed to the phloem flanks as preferred phloem loading path. Simple estimates support the pivotal role of the bundle sheath, showing that a bidirectional movement of nutrient ions and assimilates is feasible and emphasising the bundle sheath's role in nutrient and assimilate exchange.

Robust agricultural yields require consistent flower production throughout fluctuating environmental conditions. Floral primordia are produced in the inflorescence meristem, which contains a pool of continuously dividing stem cells. Daughter cells of these divisions either retain stem cell identity or are pushed to the SAM periphery, where they become competent to develop into floral primordia after receiving the appropriate signal. Thus, flower production is inherently linked to regulation of the stem cell pool. The plant hormone auxin promotes flower development as it progresses and has been shown to interact with the molecular pathways regulating stem cell maintenance. Here, we will summarize how auxin signaling contributes to stem cell maintenance and promotes flower development through the early phases of initiation, outgrowth, and floral fate establishment. Recent advances in this area suggest that auxin may serve as a signal that integrates stem cell maintenance and new flower production.

Water will be a major limitation to food production in the 21st century, and drought issues already prevail in many parts of the world. Finding solutions to ensure that farmers harvest profitable crops, and secure food supplies for families and feed for animals that will provide for them through to the next season are urgent necessities. The Interdrought community has been addressing this issue for almost 30 years in a series of international conferences, characterized by a multi-disciplinary approach across the domains of molecular biology, physiology, genetics, agronomy, breeding, environmental and social sciences, policy, and systems modeling. This special issue presents papers from the 7th edition of the conference, the first to be held in Africa, that paid special attention to drought in a smallholder context, adding a 'system' dimension to the crop focus from the previous Interdrought events (Varshney et al., 2018; Hammer et al., 2021).

Water scarcity is already set to be one of the main issues of the 21st century, because of competing needs between civil, industrial, and agricultural use. Agriculture is currently the largest user of water, but its share is bound to decrease as societies develop and clearly it needs to become more water efficient. Improving water use efficiency (WUE) at the plant level is important, but translating this at the farm/landscape level presents considerable challenges. As we move up from the scale of cells, organs, and plants to more integrated scales such as plots, fields, farm systems, and landscapes, other factors such as trade-offs need to be considered to try to improve WUE. These include choices of crop variety/species, farm management practices, landscape design, infrastructure development, and ecosystem functions, where human decisions matter. This review is a cross-disciplinary attempt to analyse approaches to addressing WUE at these different scales, including definitions of the metrics of analysis and consideration of trade-offs. The equations we present in this perspectives paper use similar metrics across scales to make them easier to connect and are developed to highlight which levers, at different scales, can improve WUE. We also refer to models operating at these different scales to assess WUE. While our entry point is plants and crops, we scale up the analysis of WUE to farm systems and landscapes.

Arbuscular mycorrhizal fungi (AMF) have been presumed to ameliorate crop tolerance to drought. Here, we review the role of AMF in maintaining water supply to plants from drying soils and the underlying biophysical mechanisms. We used a soil-plant hydraulic model to illustrate the impact of several AMF mechanisms on plant responses to edaphic drought. The AMF enhance the soil's capability to transport water and extend the effective root length, thereby attenuating the drop in matric potential at the root surface during soil drying. The synthesized evidence and the corresponding simulations demonstrate that symbiosis with AMF postpones the stress onset limit, which is defined as the disproportionality between transpiration rates and leaf water potentials, during soil drying. The symbiosis can thus help crops survive extended intervals of limited water availability. We also provide our perspective on future research needs and call for reconciling the dynamic changes in soil and root hydraulics in order to better understand the role of AMF in plant water relations in the face of climate changes.

We review approaches to maize breeding for improved drought tolerance during flowering and grain filling in the central and western US corn belt and place our findings in the context of results from public breeding. Here we show that after two decades of dedicated breeding efforts, the rate of crop improvement under drought increased from 6.2 g m-2 year-1 to 7.5 g m-2 year-1, closing the genetic gain gap with respect to the 8.6 g m-2 year-1 observed under water-sufficient conditions. The improvement relative to the long-term genetic gain was possible by harnessing favourable alleles for physiological traits available in the reference population of genotypes. Experimentation in managed stress environments that maximized the genetic correlation with target environments was key for breeders to identify and select for these alleles. We also show that the embedding of physiological understanding within genomic selection methods via crop growth models can hasten genetic gain under drought. We estimate a prediction accuracy differential (Δr) above current prediction approaches of ~30% (Δr=0.11, r=0.38), which increases with increasing complexity of the trait environment system as estimated by Shannon information theory. We propose this framework to inform breeding strategies for drought stress across geographies and crops.

Water scarcity is the primary environmental constraint affecting wheat growth and production and is increasingly exacerbated due to climatic fluctuation, which jeopardizes future food security. Most breeding efforts to improve wheat yields under drought have focused on above-ground traits. Root traits are closely associated with various drought adaptability mechanisms, but the genetic variation underlying these traits remains untapped, even though it holds tremendous potential for improving crop resilience. Here, we examined this potential by re-introducing ancestral alleles from wild emmer wheat (Triticum turgidum ssp. dicoccoides) and studied their impact on root architecture diversity under terminal drought stress. We applied an active sensing electrical resistivity tomography approach to compare a wild emmer introgression line (IL20) and its drought-sensitive recurrent parent (Svevo) under field conditions. IL20 exhibited greater root elongation under drought, which resulted in higher root water uptake from deeper soil layers. This advantage initiated at the pseudo-stem stage and increased during the transition to the reproductive stage. The increased water uptake promoted higher gas exchange rates and enhanced grain yield under drought. Overall, we show that this presumably 'lost' drought-induced mechanism of deeper rooting profile can serve as a breeding target to improve wheat productiveness under changing climate.

Adequate management of N supply, plant density, row spacing, and soil cover has proved useful for increasing grain yields and/or grain yield stability of rainfed crops over the years. We review the impact of these management practices on grain yield water-related determinants: seasonal crop evapotranspiration (ET) and water use efficiency for grain production per unit of evapotranspired water during the growing season (WUEG,ET,s). We highlight a large number of conflicting results for the impact of management on ET and expose the complexity of the ET response to environmental factors. We analyse the influence of management practices on WUEG,ET,s in terms of the three main processes controlling it: (i) the proportion of transpiration in ET (T/ET), (ii) transpiration efficiency for shoot biomass production (TEB), and (iii) the harvest index. We directly relate the impact of management practices on T/ET to their effect on crop light interception and provide evidence that management practices significantly influence TEB. To optimize WUEG,ET,s, management practices should favor soil water availability during critical periods for seed set, thereby improving the harvest index. The need to improve the performance of existing crop growth models for the prediction of water-related grain yield determinants under different management practices is also discussed.

Phytoplankton are exposed to dramatic variations in light quality when cells are carried by upwelling or downwelling currents or encounter sediment. We investigated the potential impact of light quality changes in Ostreococcus, a key marine photosynthetic picoeukaryote, by analysing changes in its transcriptome, pigment content and photophysiology after acclimation to monochromatic red, green or blue light. The clade B species RCC809, isolated from the deep euphotic zone of the tropical Atlantic Ocean, responded to blue light by accelerating cell division at the expense of storage reserves and by increasing the relative level of blue-light absorbing pigments. RCC809 responded to red and green light by increasing its potential for photoprotection. In contrast, the clade A species OTTH0595, which originates from a shallow water environment, showed no difference in photosynthetic properties and minor differences in carotenoid contents between light qualities. This was associated with the loss of candidate lightquality responsive promoter motifs identified in RCC809 genes. These results demonstrate that light quality can have a major influence on the physiology of eukaryotic phytoplankton and suggest that different light quality environments can drive selection for diverse patterns of responsiveness and environmental niche partitioning.

The water deficit experienced by crops is a function of atmospheric water demand (vapor pressure deficit) and soil water supply over the whole crop cycle. We summarize typical transpiration response patterns to soil and atmospheric drying and the sensitivity to plant hydraulic traits. We explain the transpiration response patterns using a soil-plant hydraulic framework. In both cases of drying, stomatal closure is triggered by limitations in soil-plant hydraulic conductance. However, traits impacting the transpiration response differ between the two drying processes and act at different time scales. A low plant hydraulic conductance triggers an earlier restriction in transpiration during increasing vapor pressure deficit. During soil drying, the impact of the plant hydraulic conductance is less obvious. It is rather a decrease in the belowground hydraulic conductance (related to soil hydraulic properties and root length density) that is involved in transpiration down-regulation. The transpiration response to increasing vapor pressure deficit has a daily time scale. In the case of soil drying, it acts on a seasonal scale. Varieties that are conservative in water use on a daily scale may not be conservative over longer time scales (e.g. during soil drying). This potential independence of strategies needs to be considered in environment-specific breeding for yield-based drought tolerance.

Lentil (Lens culinaris Medik.) is commonly grown in drought-prone areas where terminal heat and drought are frequent. The limited-transpiration (TRlim) trait under high vapor pressure deficit (VPD) could be a way to conserve water and increase yield under water deficit conditions. The TRlim trait was examined in cultivated and wild lentil species together with its evolution throughout the breeding pipeline. Sixty-one accessions representing the six wild lentil species (L. orientalis, L. tomentosus, L. odemensis, L. lamottei, L. ervoides, and L. nigricans) and 13 interspecific advanced lines were evaluated in their transpiration response to high VPD. A large variation in transpiration rate (TR) response to increased VPD was recorded among wild lentil accessions, with 43 accessions exhibiting a breakpoint (BP) in their TR response to increasing VPD, with values ranging from 0.92 kPa to 3.38 kPa under greenhouse conditions. Ten genotypes for the interspecific advanced lines displayed a BP with an average of 1.95 kPa, much lower than previously reported for cultivated lentil. Results from field experiments suggest that the TRlim trait (BP=0.97 kPa) positively affected yield and yield-related parameters during the years with late-season water stress. The selection of TRlim genotypes for high VPD environments could improve lentil productivity in drought-prone areas.

Under normal conditions, tight stomatal regulation of grapevines (Vitis vinifera) avoids xylem embolism . The current study evaluated grapevine responses to challenging scenarios that might lead to leaf embolism and consequential leaf damage. We hypothesized that embolism would occur if the vines experienced low xylem water potential (Ψx) shortly after bud break or later in the season under a combination of extreme drought and heat. We subjected vines to two potentially dangerous environments: 1) withholding irrigation from a vineyard grown in a heat wave-prone environment, and 2) subjecting potted vines to terminal drought one month after bud break. In the field experiment, a heat wave at the beginning of August resulted in leaf temperatures over 45°C. Still, effective stomatal response maintained the xylem water potential (Ψx) well above the embolism threshold, and no leaf desiccation was observed. In the pot experiment, leaves of well-watered vines in May were relatively vulnerable to embolism with 50% embolism (P50) at -1.8 MPa. However, when exposed to drought, these leaves acclimated their leaf P50 by 0.65 MPa in less than a week and before reaching embolism values. When dried to embolizing Ψx, the leaf damage proportion matched (percentage-wise) the leaf embolism level. Our findings indicate that embolism and leaf damage are usually avoided by the grapevines' efficient stomatal regulation and rapid acclimation of their xylem vulnerability.