The combination of a microbial and a non-microbial biostimulant increases yield in lettuce (Lactuca sativa) under salt stress conditions by up-regulating cytokinin biosynthesis.

Salinization poses a significant challenge in agriculture, exacerbated by anthropogenic global warming. Biostimulants, derived from living microorganisms or natural extracts, have emerged as valuable tools for conventional and organic agriculture. However, our understanding of the molecular mechanisms underlying the effects of biostimulants is very limited, especially in crops under real cultivation conditions. In this study, we adopted an integrative approach to investigate the effectiveness of the combined application of plant growth-promoting bacterium (Bacillus megaterium strain BM08) and a non-microbial biostimulant under control conditions (normal watering) and salt stress. After confirming the yield increase under both conditions, we investigated the molecular mechanisms underlying the observed effect by measuring a number of physiological parameters (i.e., lipid peroxidation, antioxidants, chlorophylls, total phenolics and phytohormone content), as well as RNA sequencing and primary metabolite analyses. Our findings reveal that the combined effect of the microbial and non-microbial biostimulants led to a decrease in the antioxidant response and an up-regulation of genes involved in cytokinin biosynthesis under salt stress conditions. This, in turn, resulted in a higher concentration of the bioactive cytokinin, isopentenyladenosine, in roots and leaves and an increase in γ-aminobutyric acid, a non-proteic amino acid related to abiotic stress responses. In addition, we observed a decrease in malic acid, along with an abscisic acid (ABA)-independent up-regulation of SR-kinases, a family of protein kinases associated with abiotic stress responses. Furthermore, we observed that the single application of the non-microbial biostimulant triggers an ABA-dependent response under salt stress; however, when combined with the microbial biostimulant, it potentiated the mechanisms triggered by the BM08 bacterial strain. This comprehensive investigation shows that the combination of two biostimulants is able to elicit a cytokinin-dependent response that may explain the observed yield increase under salt stress conditions.

PIF transcriptional regulators are required for rhythmic stomatal movements.

Stomata govern the gaseous exchange between the leaf and the external atmosphere, and their function is essential for photosynthesis and the global carbon and oxygen cycles. Rhythmic stomata movements in daily dark/light cycles prevent water loss at night and allow CO2 uptake during the day. How the actors involved are transcriptionally regulated and how this might contribute to rhythmicity is largely unknown. Here, we show that morning stomata opening depends on the previous night period. The transcription factors PHYTOCHROME-INTERACTING FACTORS (PIFs) accumulate at the end of the night and directly induce the guard cell-specific K+ channel KAT1. Remarkably, PIFs and KAT1 are required for blue light-induced stomata opening. Together, our data establish a molecular framework for daily rhythmic stomatal movements under well-watered conditions, whereby PIFs are required for accumulation of KAT1 at night, which upon activation by blue light in the morning leads to the K+ intake driving stomata opening.

Maternal Embryo Effect Arrest 31 (MEE31) is a moonlighting protein involved in GDP-D-mannose biosynthesis and KAT1 potassium channel regulation.

Due to anthropogenic global warming, droughts are expected to increase and water availability to decrease in the coming decades. For this reason, research is increasingly focused on developing plant varieties and crop cultivars with reduced water consumption. Transpiration occurs through stomatal pores, resulting in water loss. Potassium plays a significant role in stomatal regulation. KAT1 is an inward-rectifying potassium channel that contributes to stomatal opening. Using a yeast high-throughput screening of an Arabidopsis cDNA library, MEE31 was found to physically interact with KAT1. MEE31 was initially identified in a screen for mutants with delayed embryonic development. The gene encodes a conserved phosphomannose isomerase (PMI). We report here that MEE31 interacts with and increases KAT1 activity in yeast and this interaction was also confirmed in plants. In addition, MEE31 complements the function of the yeast homologue, whereas the truncated version recovered in the screening does not, thus uncoupling the enzymatic activity from KAT1 regulation. We show that MEE31 overexpression leads to increased stomatal opening in Arabidopsis transgenic lines. Our data suggest that MEE31 is a moonlighting protein involved in both GDP-D-mannose biosynthesis and KAT1 regulation.

The Biostimulant, Potassium Humate Ameliorates Abiotic Stress in Arabidopsis thaliana by Increasing Starch Availability.

Potassium humate is a widely used biostimulant known for its ability to enhance growth and improve tolerance to abiotic stress. However, the molecular mechanisms explaining its effects remain poorly understood. In this study, we investigated the mechanism of action of potassium humate using the model plant Arabidopsis thaliana. We demonstrated that a formulation of potassium humate effectively increased the fresh weight accumulation of Arabidopsis plants under normal conditions, salt stress (sodium or lithium chloride), and particularly under osmotic stress (mannitol). Interestingly, plants treated with potassium humate exhibited a reduced antioxidant response and lower proline accumulation, while maintaining photosynthetic activity under stress conditions. The observed sodium and osmotic tolerance induced by humate was not accompanied by increased potassium accumulation. Additionally, metabolomic analysis revealed that potassium humate increased maltose levels under control conditions but decreased levels of fructose. However, under stress, both maltose and glucose levels decreased, suggesting changes in starch utilization and an increase in glycolysis. Starch concentration measurements in leaves showed that plants treated with potassium humate accumulated less starch under control conditions, while under stress, they accumulated starch to levels similar to or higher than control plants. Taken together, our findings suggest that the molecular mechanism underlying the abiotic stress tolerance conferred by potassium humate involves its ability to alter starch content under normal growth conditions and under salt or osmotic stress.

Modulation of potassium transport to increase abiotic stress tolerance in plants.

Potassium is the major cation responsible for the maintenance of the ionic environment in plant cells. Stable potassium homeostasis is indispensable for virtually all cellular functions, and, concomitantly, viability. Plants must cope with environmental changes such as salt or drought that can alter ionic homeostasis. Potassium fluxes are required to regulate the essential process of transpiration, so a constraint on potassium transport may also affect the plant's response to heat, cold, or oxidative stress. Sequencing data and functional analyses have defined the potassium channels and transporters present in the genomes of different species, so we know most of the proteins directly participating in potassium homeostasis. The still unanswered questions are how these proteins are regulated and the nature of potential cross-talk with other signaling pathways controlling growth, development, and stress responses. As we gain knowledge regarding the molecular mechanisms underlying regulation of potassium homeostasis in plants, we can take advantage of this information to increase the efficiency of potassium transport and generate plants with enhanced tolerance to abiotic stress through genetic engineering or new breeding techniques. Here, we review current knowledge of how modifying genes related to potassium homeostasis in plants affect abiotic stress tolerance at the whole plant level.

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi.

Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, ready-to-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciens-mediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA, P7760, Pef1α, and PafpB constitutive promoters, and PglaA, PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.

Use of Yucca (Yucca schidigera) Extracts as Biostimulants to Promote Germination and Early Vigor and as Natural Fungicides.

Climate change is increasing drought and salinity in many cultivated areas, therefore threatening food production. There is a great demand for novel agricultural inputs able to maintain yield under the conditions imposed by the anthropogenic global warming. Biostimulants have been proposed as a useful tool to achieve this objective. We have investigated the biostimulant effect of different yucca (Yucca schidigera) extracts on plant growth at different stages of development under different abiotic stress conditions. The extracts were tested in the model plant Arabidopsis thaliana, and in three different crops; tomato (Solanum lycopersicum var microtom), broccoli (Brassica oleracea var. italica) and lettuce (Lactuca sativa var romana). We have found that the investigated extracts are able to promote germination and early vigor under drought/osmotic and salt stress induced either by sodium chloride or lithium chloride. This effect is particularly strong in Arabidopsis thaliana and in the Brassicaceae broccoli. We have also determined using antibiograms against the model yeast Saccharomyces cerevisiae that the evaluated extracts may be used also as a natural fungicide. The results in this report show that yucca extracts may be used to enhance early vigor in some crops and as a natural fungicide, providing a new and useful tool for farmers.

Identification of Distinctive Primary Metabolites Influencing Broccoli (Brassica oleracea, var. Italica) Taste.

Broccoli (Brassica oleracea L. var. Italica Plenck) is a cruciferous crop that is considered to be a good source of micronutrients. Better taste is a main objective for breeding, as consumers are demanding novel cultivars suited for a healthy diet, but ones that are more palatable. This study aimed to identify primary metabolites related to cultivars with better taste according to a consumer panel. For this purpose, we performed a complete primary metabolomic profile of 20 different broccoli cultivars grown in the field and contrasted the obtained data with the results of a consumer panel which evaluated the taste of the same raw buds. A statistical analysis was conducted to find primary metabolites correlating with better score in the taste panels. According to our results, sugar content is not a distinctive factor for taste in broccoli. The accumulation of the amino acids leucine, lysine and alanine, together with Myo-inositol, negatively affected taste, while a high content of γ-aminobutyric acid (GABA) is a distinctive trait for cultivars scoring high in the consumer panels. A Principal Component Analysis (PCA) allowed us to define three different groups according to the metabolomic profile of the 20 broccoli cultivars studied. Our results suggest molecular traits that could be useful as distinctive markers to predict better taste in broccoli or to design novel biotechnological or classical breeding strategies for improving broccoli taste.

A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes.

Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein-protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators. We verified a subset of 25 interactions in planta by bimolecular fluorescence complementation assays. We then constructed and analyzed a network comprising 399 TuMV-A. thaliana interactions together with intravirus and intrahost connections. In particular, we found that the host proteins targeted by TuMV are enriched in different aspects of plant responses to infections, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. The proviral or antiviral role of ten host proteins was validated by characterizing the infection dynamics in the corresponding mutant plants, supporting a proviral role for the transcriptional regulator TGA1. Comparison with similar studies with animal viruses, highlights shared fundamental features in their mode of action.

A fast method to evaluate in a combinatorial manner the synergistic effect of different biostimulants for promoting growth or tolerance against abiotic stress.

BACKGROUND: According to the most popular definition, a biostimulant is any substance or microorganism applied to plants with the aim to enhance nutrition efficiency, abiotic stress tolerance and/or crop quality traits, regardless of its nutrient content. Therefore, a biostimulant can help crops to withstand abiotic stress, while maintaining or even increasing productivity. We have previously designed a sequential system, based on two different model organisms, the baker's yeast Saccharomyces cerevisiae and the plant Arabidopsis thaliana, to evaluate the potential of different natural extracts as biostimulants employing a blind-test strategy. RESULTS: In this report, we further expand this concept to evaluate different biostimulants in a combinatorial approach to reveal the potential additive, synergistic or antagonistic effects of different combinations of biostimulants in order to design new formulations with enhanced effects on plant growth or tolerance to abiotic stress. The method is based on yeast assays (growth tests in solid medium, and continuous growth in liquid cultures) and plant assays (mass accumulation in hydroponic culture) to assess effects on early growth. CONCLUSIONS: With this novel approach, we have designed new formulations and quantified the ability to enhance growth and promote biomass accumulation under normal conditions and in the presence of abiotic stresses, such as drought, salinity or cold. This method enables a fast screen of many different products in a combinatorial manner, in order to design novel formulations of natural extracts with biostimulant potential.

Seed coat lignification level is crucial in Capsicum spp seed longevity.

Capsicum (pepper) is known for its poor seed germination, particularly seed longevity is usually much shorter than other Solanaceae. However, the molecular mechanisms involved are mostly unknown in these species. The present study examines the differences in seed longevity among Capsicum species and varietal types. Feral or less domesticated species, such as Capsicum chinense and particularly Capsicum frutescens, showed higher germination rates than the more domesticated Capsicum annuum after accelerated seed aging treatments. In addition, variability was detected in the expression of genes involved in the response to seed deterioration. The differences observed in ASPG1 expression led us to study the seed protein profile in dry and germinating seeds. Seed storage protein mobilization during germination was faster in seed aging-resistant genotypes. Similarly, the transcriptional change observed for the orthologous gene of the trans-species regulator AtHB25 prompted us to study the structure and molecular components of the seed coat in peppers. All the Capsicum pepper accessions analyzed presented very lignified testa and we observed a positive correlation between the amount of lignin and seed viability. Our results provide essential information to explain the poor germination observed in pepper seeds and provide an experimental framework for future improvements in this important character.

Distinctive Traits for Drought and Salt Stress Tolerance in Melon (Cucumis melo L.).

Melon (Cucumis melo L.) is a crop with important agronomic interest worldwide. Because of the increase of drought and salinity in many cultivation areas as a result of anthropogenic global warming, the obtention of varieties tolerant to these conditions is a major objective for agronomical improvement. The identification of the limiting factors for stress tolerance could help to define the objectives and the traits which could be improved by classical breeding or other techniques. With this objective, we have characterized, at the physiological and biochemical levels, two different cultivars (sensitive or tolerant) of two different melon varieties (Galia and Piel de Sapo) under controlled drought or salt stress. We have performed physiological measurements, a complete amino acid profile and we have determined the sodium, potassium and hormone concentrations. This has allowed us to determine that the distinctive general trait for salt tolerance in melon are the levels of phenylalanine, histidine, proline and the Na+/K+ ratio, while the distinctive traits for drought tolerance are the hydric potential, isoleucine, glycine, phenylalanine, tryptophan, serine, and asparagine. These could be useful markers for breeding strategies or to predict which varieties are likely perform better under drought or salt stress. Our study has also allowed us to identify which metabolites and physiological traits are differentially regulated upon salt and drought stress between different varieties.

Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica).

BACKGROUND: Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. RESULTS: This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. CONCLUSIONS: This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.

Physiological and Molecular Characterization of the Differential Response of Broccoli (Brassica oleracea var. Italica) Cultivars Reveals Limiting Factors for Broccoli Tolerance to Drought Stress.

Broccoli is a cruciferous crop rich in health-promoting metabolites. Due to several factors, including anthropogenic global warming, aridity is increasing in many cultivation areas. There is a great demand to characterize the drought response of broccoli and use this knowledge to develop new cultivars able to maintain yield under water constraints. The aim of this study is to characterize the drought response at the physiological and molecular level of different broccoli (Brassica oleracea L. var. Italica Plenck) cultivars, previously characterized as drought-sensitive or drought-tolerant. This approach aims to identify different traits, which can constitute limiting factors for drought stress tolerance in broccoli. For this purpose, we have compared several physiological parameters and the complete profiles of amino acids, primary metabolites, hormones, and ions of drought-tolerant and drought-sensitive cultivars under stress and control conditions. We have found that drought-tolerant cultivars presented higher levels of methionine and abscisic acid and lower amounts of urea, quinic acid, and the gluconic acid lactone. Interestingly, we have also found that a drought treatment increases the levels of most essential amino acids in leaves and in florets. Our results have established physiological and molecular traits useful as distinctive markers to predict drought tolerance in broccoli or which could be reliably used for breeding new cultivars adapted to water scarcity. We have also found that a drought treatment increases the content of essential amino acids in broccoli.

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond.

Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate-based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host-specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi-a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal-specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium-mediated transformation. We also describe the use of a set of web-based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Software-assisted modular DNA assembly of a two gene expression-cassette with GB Basic Protocol 2: Agrobacterium tumefaciens-mediated transformation of filamentous fungi Basic Protocol 3: Software-assisted modular DNA assembly of a gene disruption-cassette using GB Basic Protocol 4: Obtaining disruption transformants.

BCL2-ASSOCIATED ATHANOGENE4 Regulates the KAT1 Potassium Channel and Controls Stomatal Movement.

Potassium (K+) is a key monovalent cation necessary for multiple aspects of cell growth and survival. In plants, this cation also plays a key role in the control of stomatal movement. KAT1 and its homolog KAT2 are the main inward rectifying channels present in guard cells, mediating K+ influx into these cells, resulting in stomatal opening. To gain further insight into the regulation of these channels, we performed a split-ubiquitin protein-protein interaction screen searching for KAT1 interactors in Arabidopsis (Arabidopsis thaliana). We characterized one of these candidates, BCL2-ASSOCIATED ATHANOGENE4 (BAG4), in detail using biochemical and genetic approaches to confirm this interaction and its effect on KAT1 activity. We show that BAG4 improves KAT1-mediated K+ transport in two heterologous systems and provide evidence that in plants, BAG4 interacts with KAT1 and favors the arrival of KAT1 at the plasma membrane. Importantly, lines lacking or overexpressing the BAG4 gene show altered KAT1 plasma membrane accumulation and alterations in stomatal movement. Our data allowed us to identify a KAT1 regulator and define a potential target for the plant BAG family. The identification of physiologically relevant regulators of K+ channels will aid in the design of approaches that may impact drought tolerance and pathogen susceptibility.

Saccharomyces cerevisiae as a Tool to Investigate Plant Potassium and Sodium Transporters.

Sodium and potassium are two alkali cations abundant in the biosphere. Potassium is essential for plants and its concentration must be maintained at approximately 150 mM in the plant cell cytoplasm including under circumstances where its concentration is much lower in soil. On the other hand, sodium must be extruded from the plant or accumulated either in the vacuole or in specific plant structures. Maintaining a high intracellular K+/Na+ ratio under adverse environmental conditions or in the presence of salt is essential to maintain cellular homeostasis and to avoid toxicity. The baker's yeast, Saccharomyces cerevisiae, has been used to identify and characterize participants in potassium and sodium homeostasis in plants for many years. Its utility resides in the fact that the electric gradient across the membrane and the vacuoles is similar to plants. Most plant proteins can be expressed in yeast and are functional in this unicellular model system, which allows for productive structure-function studies for ion transporting proteins. Moreover, yeast can also be used as a high-throughput platform for the identification of genes that confer stress tolerance and for the study of protein-protein interactions. In this review, we summarize advances regarding potassium and sodium transport that have been discovered using the yeast model system, the state-of-the-art of the available techniques and the future directions and opportunities in this field.

FungalBraid: A GoldenBraid-based modular cloning platform for the assembly and exchange of DNA elements tailored to fungal synthetic biology.

Current challenges in the study and biotechnological exploitation of filamentous fungi are the optimization of DNA cloning and fungal genetic transformation beyond model fungi, the open exchange of ready-to-use and standardized genetic elements among the research community, and the availability of universal synthetic biology tools and rules. The GoldenBraid (GB) cloning framework is a Golden Gate-based DNA cloning system developed for plant synthetic biology through Agrobacterium tumefaciens-mediated genetic transformation (ATMT). In this study, we develop reagents for the adaptation of GB version 3.0 from plants to filamentous fungi through: (i) the expansion of the GB toolbox with the domestication of fungal-specific genetic elements; (ii) the design of fungal-specific GB structures; and (iii) the ATMT and gene disruption of the plant pathogen Penicillium digitatum as a proof of concept. Genetic elements domesticated into the GB entry vector pUPD2 include promoters, positive and negative selection markers and terminators. Interestingly, some GB elements can be directly exchanged between plants and fungi, as demonstrated with the marker hph for HygR or the fluorescent protein reporter YFP. The iterative modular assembly of elements generates an endless number of diverse transcriptional units and other higher order combinations in the pDGB3α/pDGB3Ω destination vectors. Furthermore, the original plant GB syntax was adapted here to incorporate specific GB structures for gene disruption through homologous recombination and dual selection. We therefore have successfully adapted the GB technology for the ATMT of fungi. We propose the name of FungalBraid (FB) for this new branch of the GB technology that provides open, exchangeable and collaborative resources to the fungal research community.