Transcriptome, Biochemical and Phenotypic Analysis of the Effects of a Precision Engineered Biostimulant for Inducing Salinity Stress Tolerance in Tomato.

Salinity stress is a major problem affecting plant growth and crop productivity. While plant biostimulants have been reported to be an effective solution to tackle salinity stress in different crops, the key genes and metabolic pathways involved in these tolerance processes remain unclear. This study focused on integrating phenotypic, physiological, biochemical and transcriptome data obtained from different tissues of Solanum lycopersicum L. plants (cv. Micro-Tom) subjected to a saline irrigation water program for 61 days (EC: 5.8 dS/m) and treated with a combination of protein hydrolysate and Ascophyllum nodosum-derived biostimulant, namely PSI-475. The biostimulant application was associated with the maintenance of higher K+/Na+ ratios in both young leaf and root tissue and the overexpression of transporter genes related to ion homeostasis (e.g., NHX4, HKT1;2). A more efficient osmotic adjustment was characterized by a significant increase in relative water content (RWC), which most likely was associated with osmolyte accumulation and upregulation of genes related to aquaporins (e.g., PIP2.1, TIP2.1). A higher content of photosynthetic pigments (+19.8% to +27.5%), increased expression of genes involved in photosynthetic efficiency and chlorophyll biosynthesis (e.g., LHC, PORC) and enhanced primary carbon and nitrogen metabolic mechanisms were observed, leading to a higher fruit yield and fruit number (47.5% and 32.5%, respectively). Overall, it can be concluded that the precision engineered PSI-475 biostimulant can provide long-term protective effects on salinity stressed tomato plants through a well-defined mode of action in different plant tissues.

Investigation of the direct effect of a precision Ascophyllum nodosum biostimulant on nitrogen use efficiency in wheat seedlings.

Reduction in the greenhouse gas (GHG) emissions and nitrogen (N) pollution of ground water by improving nitrogen use efficiency (NUE) in crops has become an intensively investigated research topic in pursuit of a more sustainable future. Although, distinct solutions have been proposed there are only a few reports documenting the detailed interplay between observed plant growth dynamics and changes in plant N related transcriptional and biochemical changes. It was previously demonstrated that the application of a formulated biostimulant (PSI-362) derived from Ascophyllum nodosum (ANE) improves N uptake in Arabidopsis thaliana and in barley. In this study, the effect of PSI-362 on the growth dynamics of wheat seedlings was evaluated at different biostimulant and N supplementation rates. Wheat grown on N deficient MS medium was also analysed from the first hour of the treatment until the depletion of the nutrients in the medium 9 days later. During this time the biomass increase measured for PSI-362 treated plants versus untreated controls was associated with increased nitrate uptake, with surplus N assimilated by the biomass in the form of glutamate, glutamine, free amino acids, soluble proteins, and chlorophyll. Phenotypical and biochemical analysis were supported by evaluation of differential expression of genetic markers involved in nitrate perception and transport (TaNRT1.1/NPF6.3), nitrate and nitrite reduction (TaNR1 and TaNiR1) and assimilation (TaGDH2, TaGoGAT, TaGS1). Finally, a comparative analysis of the precision biostimulant PSI-362 and two generic ANEs demonstrated that the NUE effect greatly differs depending on the ANE formulation used.

Reducing Nitrogen Input in Barley Crops While Maintaining Yields Using an Engineered Biostimulant Derived From Ascophyllum nodosum to Enhance Nitrogen Use Efficiency.

Intensive agricultural production utilizes large amounts of nitrogen (N) mineral fertilizers that are applied to the soil to secure high crop yields. Unfortunately, up to 65% of this N fertilizer is not taken up by crops and is lost to the environment. To compensate these issues, growers usually apply more fertilizer than crops actually need, contributing significantly to N pollution and to GHG emissions. In order to combat the need for such large N inputs, a better understanding of nitrogen use efficiency (NUE) and agronomic solutions that increase NUE within crops is required. The application of biostimulants derived from extracts of the brown seaweed Ascophyllum nodosum has long been accepted by growers as a sustainable crop production input. However, little is known on how Ascophyllum nodosum extracts (ANEs) can influence mechanisms of N uptake and assimilation in crops to allow reduced N application. In this work, a significant increase in nitrate accumulation in Arabidopsis thaliana 6 days after applying the novel proprietary biostimulant PSI-362 was observed. Follow-up studies in barley crops revealed that PSI-362 increases NUE by 29.85-60.26% under 75% N input in multi-year field trials. When PSI-362 was incorporated as a coating to the granular N fertilizer calcium ammonium nitrate and applied to barley crop, a coordinated stimulation of N uptake and assimilation markers was observed. A key indicator of biostimulant performance was increased nitrate content in barley shoot tissue 22 days after N fertilizer application (+17.9-72.2%), that was associated with gene upregulation of root nitrate transporters (NRT1.1, NRT2.1, and NRT1.5). Simultaneously, PSI-362 coated fertilizer enhanced nitrate reductase and glutamine synthase activities, while higher content of free amino acids, soluble protein and photosynthetic pigments was measured. These biological changes at stem elongation stage were later translated into enhanced NUE traits in harvested grain. Overall, our results support the agronomic use of this engineered ANE that allowed a reduction in N fertilizer usage while maintaining or increasing crop yield. The data suggests that it can be part of the solution for the successful implementation of mitigation policies for water quality and GHG emissions from N fertilizer usage.

Ascophyllum nodosum Extract (SealicitTM) Boosts Soybean Yield Through Reduction of Pod Shattering-Related Seed Loss and Enhanced Seed Production.

Soybean is one of the most valuable commercial crops because of its high protein, carbohydrate, and oil content. The land area cultivated with soybean in subtropical regions, such as Brazil, is continuously expanding, in some instances at the expense of carbon storing natural habitats. Strategies to decrease yield/seed losses and increase production efficiency are urgently required to meet global demand for soybean in a sustainable manner. Here, we evaluated the effectiveness of an Ascophyllum nodosum extract (ANE), SealicitTM, in increasing yields of different soybean varieties, in two geographical regions (Canada and Brazil). In addition, we investigated the potential of SealicitTM to reduce pod shattering at the trials in Brazil. Three different concentrations of SealicitTM were applied to pod shatter-susceptible (SS) UFUS 6901 and shatter-resistant (SR) UFUS 7415 varieties to assess their impact on pod firmness. SS variety demonstrated a significant decrease in pod shattering, which coincided with deregulation of GmPDH1.1 and GmSHAT1-5 expression, genes that determine pod dehiscence, and higher seed weight per pod. SealicitTM application to the SR variety did not significantly alter its inherent pod shatter resistance, but provided higher increases in seed yield at harvest. This yield increase maybe associated with to other yield components stimulated by the biostimulant. This work demonstrates that SealicitTM, which has previously been shown to improve pod firmness in Arabidopsis and selected commercial oilseed rape varieties through IND gene down-regulation, also has the potential to improve pod resistance and seed productivity in soybean, a member of the legume family sharing a similar strategy for seed dispersal.

HEARTBREAK Controls Post-translational Modification of INDEHISCENT to Regulate Fruit Morphology in Capsella.

Morphological variation is the basis of natural diversity and adaptation. For example, angiosperms (flowering plants) evolved during the Cretaceous period more than 100 mya and quickly colonized terrestrial habitats [1]. A major reason for their astonishing success was the formation of fruits, which exist in a myriad of different shapes and sizes [2]. Evolution of organ shape is fueled by variation in expression patterns of regulatory genes causing changes in anisotropic cell expansion and division patterns [3-5]. However, the molecular mechanisms that alter the polarity of growth to generate novel shapes are largely unknown. The heart-shaped fruits produced by members of the Capsella genus comprise an anatomical novelty, making it particularly well suited for studies on morphological diversification [6-8]. Here, we show that post-translational modification of regulatory proteins provides a critical step in organ-shape formation. Our data reveal that the SUMO protease, HEARTBREAK (HTB), from Capsella rubella controls the activity of the key regulator of fruit development, INDEHISCENT (CrIND in C. rubella), via de-SUMOylation. This post-translational modification initiates a transduction pathway required to ensure precisely localized auxin biosynthesis, thereby facilitating anisotropic cell expansion to ultimately form the heart-shaped Capsella fruit. Therefore, although variation in the expression of key regulatory genes is known to be a primary driver in morphological evolution, our work demonstrates how other processes-such as post-translational modification of one such regulator-affects organ morphology.

Ascophyllum nodosum Extract Biostimulant Processing and Its Impact on Enhancing Heat Stress Tolerance During Tomato Fruit Set.

The application of biostimulants derived from extracts of the brown seaweed Ascophyllum nodosum has long been accepted by growers to have productivity benefits in stressed crops. The impact of the processing method of the A. nodosum biomass is also known to affect compositional and physicochemical properties. However, the identification of the mechanisms by which processing parameters of Ascophyllum nodosum extracts (ANEs) affect biostimulant performance in abiotically stressed crops is still poorly understood. In this study, we performed a comparative analysis of two carbohydrate-rich formulations derived from A. nodosum: C129, an ANE obtained at low temperatures through a gentle extraction and the novel proprietary PSI-494 extracted under high temperatures and alkaline conditions. We tested the efficiency of both ANEs in unstressed conditions as well as in mitigating long-term moderate heat stress in tomato (Lycopersicon esculentum, cv. Micro Tom) during the reproductive stage. Both ANEs showed significant effects on flower development, pollen viability, and fruit production in both conditions. However, PSI-494 significantly surpassed the heat stress tolerance effect of C129, increasing fruit number by 86% compared to untreated plants growing under heat stress conditions. The variation in efficacy was associated with different molecular mass distribution profiles of the ANEs. Specific biochemical and transcriptional changes were observed with enhanced thermotolerance. PSI-494 was characterized as an ANE formulation with lower molecular weight constituents, which was associated with an accumulation of soluble sugars, and gene transcription of protective heat shock proteins (HSPs) in heat stressed tomato flowers before fertilization. These findings suggest that specialized ANE biostimulants targeting the negative effects of periods of heat stress during the important reproductive stage can lead to significant productivity gains.

A plant biostimulant from the seaweed Ascophyllum nodosum (Sealicit) reduces podshatter and yield loss in oilseed rape through modulation of IND expression.

The yield of podded crops such as oilseed rape (OSR) is limited by evolutionary adaptations of the plants for more efficient and successful seed dispersal for survival. These plants have evolved dehiscent dry fruits that shatter along a specifically developed junction at carpel margins. A number of strategies such as pod sealants, GMOs and hybrids have been developed to mitigate the impact of pod shatter on crop yield with limited success. Plant biostimulants have been shown to influence plant development. A challenge in plant biostimulant research is elucidating the mechanisms of action. Here we have focused on understanding the effect of an Ascophyllum nodosum based biostimulant (Sealicit) on fruit development and seed dispersal trait in Arabidopsis and OSR at genetic and physiological level. The results indicate that Sealicit is affecting the expression of the major regulator of pod shattering, INDEHISCENT, as well as disrupting the auxin minimum. Both factors influence the formation of the dehiscence zone and consequently reduce pod shattering. Unravelling the mode of action of this unique biostimulant provides data to support its effectiveness in reducing pod shatter and highlights its potential for growers to increase seed yield in a number of OSR varieties.

Regulatory Diversification of INDEHISCENT in the Capsella Genus Directs Variation in Fruit Morphology.

Evolution of gene-regulatory sequences is considered the primary driver of morphological variation [1-3]. In animals, the diversity of body plans between distantly related phyla is due to the differential expression patterns of conserved "toolkit" genes [4]. In plants, variation in expression domains similarly underlie most of the reported diversity of organ shape both in natural evolution and in the domestication of crops [5-9]. The heart-shaped fruit from members of the Capsella genus is a morphological novelty that has evolved after Capsella diverged from Arabidopsis ∼8 mya [10]. Comparative studies of fruit growth in Capsella and Arabidopsis revealed that the difference in shape is caused by local control of anisotropic growth [11]. Here, we show that sequence variation in regulatory domains of the fruit-tissue identity gene, INDEHISCENT (IND), is responsible for expansion of its expression domain in the heart-shaped fruits from Capsella rubella. We demonstrate that expression of this CrIND gene in the apical part of the valves in Capsella contributes to the heart-shaped appearance. While studies on morphological diversity have revealed the importance of cis-regulatory sequence evolution, few examples exist where the downstream effects of such variation have been characterized in detail. We describe here how CrIND exerts its function on Capsella fruit shape by binding sequence elements of auxin biosynthesis genes to activate their expression and ensure auxin accumulation into highly localized maxima in the fruit valves. Thus, our data provide a direct link between changes in expression pattern and altered hormone homeostasis in the evolution of morphological novelty.

Fruit shape diversity in the Brassicaceae is generated by varying patterns of anisotropy.

Fruits exhibit a vast array of different 3D shapes, from simple spheres and cylinders to more complex curved forms; however, the mechanism by which growth is oriented and coordinated to generate this diversity of forms is unclear. Here, we compare the growth patterns and orientations for two very different fruit shapes in the Brassicaceae: the heart-shaped Capsella rubella silicle and the near-cylindrical Arabidopsis thaliana silique. We show, through a combination of clonal and morphological analyses, that the different shapes involve different patterns of anisotropic growth during three phases. These experimental data can be accounted for by a tissue-level model in which specified growth rates vary in space and time and are oriented by a proximodistal polarity field. The resulting tissue conflicts lead to deformation of the tissue as it grows. The model allows us to identify tissue-specific and temporally specific activities required to obtain the individual shapes. One such activity may be provided by the valve-identity gene FRUITFULL, which we show through comparative mutant analysis to modulate fruit shape during post-fertilisation growth of both species. Simple modulations of the model presented here can also broadly account for the variety of shapes in other Brassicaceae species, thus providing a simplified framework for fruit development and shape diversity.

Cellular mechanisms for cargo delivery and polarity maintenance at different polar domains in plant cells.

The asymmetric localization of proteins in the plasma membrane domains of eukaryotic cells is a fundamental manifestation of cell polarity that is central to multicellular organization and developmental patterning. In plants, the mechanisms underlying the polar localization of cargo proteins are still largely unknown and appear to be fundamentally distinct from those operating in mammals. Here, we present a systematic, quantitative comparative analysis of the polar delivery and subcellular localization of proteins that characterize distinct polar plasma membrane domains in plant cells. The combination of microscopic analyses and computational modeling revealed a mechanistic framework common to diverse polar cargos and underlying the establishment and maintenance of apical, basal, and lateral polar domains in plant cells. This mechanism depends on the polar secretion, constitutive endocytic recycling, and restricted lateral diffusion of cargos within the plasma membrane. Moreover, our observations suggest that polar cargo distribution involves the individual protein potential to form clusters within the plasma membrane and interact with the extracellular matrix. Our observations provide insights into the shared cellular mechanisms of polar cargo delivery and polarity maintenance in plant cells.

Diversification of fruit shape in the Brassicaceae family.

KEY MESSAGE: Diversity in fruit shape. Angiosperms (flowering plants) evolved during the Cretaceous Period more than 100 million years ago and quickly colonized all terrestrial habitats on the planet. A major reason for their success was the formation of fruits that would protect and nurture the developing seeds. Moreover, a massive range of diversity in fruit shape occurred during a relatively short time, which allowed for the development of ingenious ways of fertilization as well as strategies for efficient seed dispersal. The Brassicaceae family more than any exemplifies the diversity in fruit morphologies, thus providing an ideal group of plants to study how specific shapes are established. Although many genes controlling fruit patterning in the model plant Arabidopsis thaliana have been identified, the processes of carpel and fruit morphogenesis are still poorly understood. Moreover, Arabidopsis fruits are relatively simple in their structure and are therefore not ideally suited for analyzing processes of morphology determination without comparison to species with differently shaped fruits. Here, we review the diversity of fruit shape within the Brassicaceae family. As an example we describe the close relative of Arabidopsis, Capsella rubella that develops flat, heart-shaped fruits showing and highlighting its potential as a model system for research into organ shape. Recent progress in genomics including fast and cheap genome sequencing and annotation as well as development of mutant populations has opened entirely new and exciting possibilities of studying the mechanisms and processes underlying fruit formation in angiosperms.

Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane.

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall-encapsulated plant cells. We have used super-resolution and semi-quantitative live-cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar-competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super-polar recycling. Within the plasma membrane, PINs are recruited into non-mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin-dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super-polar exocytosis have primary importance for PIN polarity maintenance.

Light-mediated polarization of the PIN3 auxin transporter for the phototropic response in Arabidopsis.

Phototropism is an adaptation response, through which plants grow towards the light. It involves light perception and asymmetric distribution of the plant hormone auxin. Here we identify a crucial part of the mechanism for phototropism, revealing how light perception initiates auxin redistribution that leads to directional growth. We show that light polarizes the cellular localization of the auxin efflux carrier PIN3 in hypocotyl endodermis cells, resulting in changes in auxin distribution and differential growth. In the dark, high expression and activity of the PINOID (PID) kinase correlates with apolar targeting of PIN3 to all cell sides. Following illumination, light represses PINOID transcription and PIN3 is polarized specifically to the inner cell sides by GNOM ARF GTPase GEF (guanine nucleotide exchange factor)-dependent trafficking. Thus, differential trafficking at the shaded and illuminated hypocotyl side aligns PIN3 polarity with the light direction, and presumably redirects auxin flow towards the shaded side, where auxin promotes growth, causing hypocotyls to bend towards the light. Our results imply that PID phosphorylation-dependent recruitment of PIN proteins into distinct trafficking pathways is a mechanism to polarize auxin fluxes in response to different environmental and endogenous cues.

Trafficking to the outer polar domain defines the root-soil interface.

In animals, the interface between organism and environment is constituted by the epithelium. In plants, the exchange of nutrients and signals between root and soil is crucial for their survival, but the cellular mechanisms underlying the epithelium-like function and specific localization of proteins to the root surface have not been identified. Here we analyze the mechanism of polar delivery to the root-soil interface of the proteins BOR4, ABCG37, and PEN3, which transport nutrients, transport plant hormones, and are required for pathogen defense, respectively. The simultaneous visualization of these proteins and the apical and basal cargos in a single cell demonstrates that the outermost cell side represents an additional polar domain. Delivery to this outer polar domain depends on ARF GEF and actin function but does not require known molecular components of the apical or basal targeting. The outer polar delivery is, in contrast to known basal and apical cargos, mediated by the polar secretion. Our findings show that the outermost cell membranes of roots define an additional polar domain in plant cells along with a specific, previously uncharacterized, polar targeting mechanism that is important for defining the functional, epithelium-like root-soil interface.

Arabidopsis PIS1 encodes the ABCG37 transporter of auxinic compounds including the auxin precursor indole-3-butyric acid.

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.

Cellular and molecular requirements for polar PIN targeting and transcytosis in plants.

The polar, sub-cellular localization of PIN auxin efflux carriers determines the direction of intercellular auxin flow, thus defining the spatial aspect of auxin signalling. Dynamic, transcytosis-like relocalizations of PIN proteins occur in response to external and internal signals, integrating these signals into changes in auxin distribution. Here, we examine the cellular and molecular mechanisms of polar PIN delivery and transcytosis. The mechanisms of the ARF-GEF-dependent polar targeting and transcytosis are well conserved and show little variations among diverse Arabidopsis ecotypes consistent with their fundamental importance in regulating plant development. At the cellular level, we refine previous findings on the role of the actin cytoskeleton in apical and basal PIN targeting, and identify a previously unknown role for microtubules, specifically in basal targeting. PIN protein delivery to different sides of the cell is mediated by ARF-dependent trafficking with a previously unknown complex level of distinct ARF-GEF vesicle trafficking regulators. Our data suggest that alternative recruitment of PIN proteins by these distinct pathways can account for cell type- and cargo-specific aspects of polar targeting, as well as for polarity changes in response to different signals. The resulting dynamic PIN positioning to different sides of cells defines a three-dimensional pattern of auxin fluxes within plant tissues.