Molecular and genetic bases of heat stress responses in crop plants and breeding for increased resilience and productivity.

To ensure the food security of future generations and to address the challenge of the 'no hunger zone' proposed by the FAO (Food and Agriculture Organization), crop production must be doubled by 2050, but environmental stresses are counteracting this goal. Heat stress in particular is affecting agricultural crops more frequently and more severely. Since the discovery of the physiological, molecular, and genetic bases of heat stress responses, cultivated plants have become the subject of intense research on how they may avoid or tolerate heat stress by either using natural genetic variation or creating new variation with DNA technologies, mutational breeding, or genome editing. This review reports current understanding of the genetic and molecular bases of heat stress in crops together with recent approaches to creating heat-tolerant varieties. Research is close to a breakthrough of global relevance, breeding plants fitter to face the biggest challenge of our time.

Development of an In Vivo Sensor to Monitor the Effects of Vapour Pressure Deficit (VPD) Changes to Improve Water Productivity in Agriculture.

Environment, biodiversity and ecosystem services are essential to ensure food security and nutrition. Managing natural resources and mainstreaming biodiversity across agriculture sectors are keys towards a sustainable agriculture focused on resource efficiency. Vapour Pressure Deficit (VPD) is considered the main driving force of water movements in the plant vascular system, however the tools available to monitor this parameter are usually based on environmental monitoring. The driving motif of this paper is the development of an in-vivo sensor to monitor the effects of VPD changes in the plant. We have used an in vivo sensor, termed "bioristor", to continuously monitor the changes occurring in the sap ion's status when plants experience different VPD conditions and we observed a specific R (sensor response) trend in response to VPD. The possibility to directly monitor the physiological changes occurring in the plant in different VPD conditions, can be used to increase efficiency of the water management in controlled conditions thus achieving a more sustainable use of natural resources.

Pyramiding PvPGIP2 and TAXI-III But Not PvPGIP2 and PMEI Enhances Resistance Against Fusarium graminearum.

Plant protein inhibitors counteract the activity of cell wall-degrading enzymes (CWDEs) secreted by pathogens to breach the plant cell-wall barrier. Transgenic plants expressing a single protein inhibitor restrict pathogen infections. However, since pathogens secrete a number of CWDEs at the onset of infection, we combined more inhibitors in a single wheat genotype to reinforce further the cell-wall barrier. We combined polygalacturonase (PG) inhibiting protein (PGIP) and pectin methyl esterase inhibitor (PMEI), both controlling the activity of PG, one of the first CWDEs secreted during infection. We also pyramided PGIP and TAXI-III, a xylanase inhibitor that controls the activity of xylanases, key factors for the degradation of xylan, a main component of cereal cell wall. We demonstrated that the pyramiding of PGIP and PMEI did not contribute to any further improvement of disease resistance. However, the presence of both pectinase inhibitors ensured a broader spectrum of disease resistance. Conversely, the PGIP and TAXI-III combination contributed to further improvement of Fusarium head blight (FHB) resistance, probably because these inhibitors target the activity of different types of CWDEs, i.e., PGs and xylanases. Worth mentioning, the reduction of FHB symptoms is accompanied by a reduction of deoxynivalenol accumulation with a foreseen great benefit to human and animal health.

Involvement of Fungal Pectin Methylesterase Activity in the Interaction Between Fusarium graminearum and Wheat.

The genome of Fusarium graminearum, the causal agent of Fusarium head blight of wheat, contains two putative pectin methylesterase (PME)-encoding genes. However, when grown in liquid culture containing pectin, F. graminearum produces only a single PME, which was purified and identified. Its encoding gene, expressed during wheat spike infection, was disrupted by targeted homologous recombination. Two Δpme mutant strains lacked PME activity but were still able to grow on highly methyl-esterified pectin even though their polygalacturonase (PG) activity showed a reduced capacity to depolymerize this substrate. The enzymatic assays performed with purified F. graminearum PG and PME demonstrated an increase in PG activity in the presence of PME on highly methyl-esterified pectin. The virulence of the mutant strains was tested on Triticum aestivum and Triticum durum spikes, and a significant reduction in the percentage of symptomatic spikelets was observed between 7 and 12 days postinfection compared with wild type, demonstrating that the F. graminearum PME contributes to fungal virulence on wheat by promoting spike colonization in the initial and middle stages of infection. In contrast, transgenic wheat plants with increased levels of pectin methyl esterification did not show any increase in resistance to the Δpme mutant, indicating that the infectivity of the fungus relies only to a certain degree on pectin degradation.

PvPGIP2 Accumulation in Specific Floral Tissues But Not in the Endosperm Limits Fusarium graminearum Infection in Wheat.

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most destructive fungal diseases of wheat worldwide. The pathogen infects the spike at flowering time and causes severe yield losses, deterioration of grain quality, and accumulation of mycotoxins. The understanding of the precise means of pathogen entry and colonization of floral tissue is crucial to providing effective protection against FHB. Polygalacturonase (PG) inhibiting proteins (PGIPs) are cell-wall proteins that inhibit the activity of PGs, a class of pectin-depolymerizing enzymes secreted by microbial pathogens, including Fusarium spp. The constitutive expression of a bean PGIP (PvPGIP2) limits FHB symptoms and reduces mycotoxin accumulation in wheat grain. To better understand which spike tissues play major roles in limiting F. graminearum infection, we explored the use of PvPGIP2 to defend specific spike tissues. We show here that the simultaneous expression of PvPGIP2 in lemma, palea, rachis, and anthers reduced FHB symptoms caused by F. graminearum compared with symptoms in infected nontransgenic plants. However, the expression of PvPGIP2 only in the endosperm did not affect FHB symptom development, indicating that once the pathogen has reached the endosperm, inhibition of the pathogen's PG activity is not effective in preventing its further spread.

Biochemical and molecular characterization of Avena indolines and their role in kernel texture.

Among cereals, Avena sativa is characterized by an extremely soft endosperm texture, which leads to some negative agronomic and technological traits. On the basis of the well-known softening effect of puroindolines in wheat kernel texture, in this study, indolines and their encoding genes are investigated in Avena species at different ploidy levels. Three novel 14 kDa proteins, showing a central hydrophobic domain with four tryptophan residues and here named vromindoline (VIN)-1,2 and 3, were identified. Each VIN protein in diploid oat species was found to be synthesized by a single Vin gene whereas, in hexaploid A. sativa, three Vin-1, three Vin-2 and two Vin-3 genes coding for VIN-1, VIN-2 and VIN-3, respectively, were described and assigned to the A, C or D genomes based on similarity to their counterparts in diploid species. Expression of oat vromindoline transgenes in the extra-hard durum wheat led to accumulation of vromindolines in the endosperm and caused an approximate 50 % reduction of grain hardness, suggesting a central role for vromindolines in causing the extra-soft texture of oat grain. Further, hexaploid oats showed three orthologous genes coding for avenoindolines A and B, with five or three tryptophan residues, respectively, but very low amounts of avenoindolines were found in mature kernels. The present results identify a novel protein family affecting cereal kernel texture and would further elucidate the phylogenetic evolution of Avena genus.

An asparagine residue at the N-terminus affects the maturation process of low molecular weight glutenin subunits of wheat endosperm.

BACKGROUND: Wheat glutenin polymers are made up of two main subunit types, the high- (HMW-GS) and low- (LMW-GS) molecular weight subunits. These latter are represented by heterogeneous proteins. The most common, based on the first amino acid of the mature sequence, are known as LMW-m and LMW-s types. The mature sequences differ as a consequence of three extra amino acids (MET-) at the N-terminus of LMW-m types. The nucleotide sequences of their encoding genes are, however, nearly identical, so that the relationship between gene and protein sequences is difficult to ascertain.It has been hypothesized that the presence of an asparagine residue in position 23 of the complete coding sequence for the LMW-s type might account for the observed three-residue shortened sequence, as a consequence of cleavage at the asparagine by an asparaginyl endopeptidase. RESULTS: We performed site-directed mutagenesis of a LMW-s gene to replace asparagine at position 23 with threonine and thus convert it to a candidate LMW-m type gene. Similarly, a candidate LMW-m type gene was mutated at position 23 to replace threonine with asparagine. Next, we produced transgenic durum wheat (cultivar Svevo) lines by introducing the mutated versions of the LMW-m and LMW-s genes, along with the wild type counterpart of the LMW-m gene.Proteomic comparisons between the transgenic and null segregant plants enabled identification of transgenic proteins by mass spectrometry analyses and Edman N-terminal sequencing. CONCLUSIONS: Our results show that the formation of LMW-s type relies on the presence of an asparagine residue close to the N-terminus generated by signal peptide cleavage, and that LMW-GS can be quantitatively processed most likely by vacuolar asparaginyl endoproteases, suggesting that those accumulated in the vacuole are not sequestered into stable aggregates that would hinder the action of proteolytic enzymes. Rather, whatever is the mechanism of glutenin polymer transport to the vacuole, the proteins remain available for proteolytic processing, and can be converted to the mature form by the removal of a short N-terminal sequence.

Kiwi 4.0: In Vivo Real-Time Monitoring to Improve Water Use Efficiency in Yellow Flesh Actinidia chinensis.

This manuscript reports the application of sensors for water use efficiency with a focus on the application of an in vivo OECT biosensor. In two distinct experimental trials, the in vivo sensor bioristor was applied in yellow kiwi plants to monitor, in real-time and continuously, the changes in the composition and concentration of the plant sap in an open field during plant growth and development. The bioristor response and physiological data, together with other fruit sensor monitoring data, were acquired and combined in both trials, giving a complete picture of the biosphere conditions. A high correlation was observed between the bioristor index (ΔIgs), the canopy cover expressed as the fraction of intercepted PAR (fi_PAR), and the soil water content (SWC). In addition, the bioristor was confirmed to be a good proxy for the occurrence of drought in kiwi plants; in fact, a period of drought stress was identified within the month of July. A novelty of the bioristor measurements was their ability to detect in advance the occurrence of defoliation, thereby reducing yield and quality losses. A plant-based irrigation protocol can be achieved and tailored based on real plant needs, increasing water use sustainability and preserving high-quality standards.

Constitutive expression of the xylanase inhibitor TAXI-III delays Fusarium head blight symptoms in durum wheat transgenic plants.

Cereals contain xylanase inhibitor (XI) proteins which inhibit microbial xylanases and are considered part of the defense mechanisms to counteract microbial pathogens. Nevertheless, in planta evidence for this role has not been reported yet. Therefore, we produced a number of transgenic plants constitutively overexpressing TAXI-III, a member of the TAXI type XI that is induced by pathogen infection. Results showed that TAXI-III endows the transgenic wheat with new inhibition capacities. We also showed that TAXI-III is correctly secreted into the apoplast and possesses the expected inhibition parameters against microbial xylanases. The new inhibition properties of the transgenic plants correlate with a significant delay of Fusarium head blight disease symptoms caused by Fusarium graminearum but do not significantly influence leaf spot symptoms caused by Bipolaris sorokiniana. We showed that this contrasting result can be due to the different capacity of TAXI-III to inhibit the xylanase activity of these two fungal pathogens. These results provide, for the first time, clear evidence in planta that XI are involved in plant defense against fungal pathogens and show the potential to manipulate TAXI-III accumulation to improve wheat resistance against F. graminearum.

Plant responses to climate change, how global warming may impact on food security: a critical review.

Global agricultural production must double by 2050 to meet the demands of an increasing world human population but this challenge is further exacerbated by climate change. Environmental stress, heat, and drought are key drivers in food security and strongly impacts on crop productivity. Moreover, global warming is threatening the survival of many species including those which we rely on for food production, forcing migration of cultivation areas with further impoverishing of the environment and of the genetic variability of crop species with fall out effects on food security. This review considers the relationship of climatic changes and their bearing on sustainability of natural and agricultural ecosystems, as well as the role of omics-technologies, genomics, proteomics, metabolomics, phenomics and ionomics. The use of resource saving technologies such as precision agriculture and new fertilization technologies are discussed with a focus on their use in breeding plants with higher tolerance and adaptability and as mitigation tools for global warming and climate changes. Nevertheless, plants are exposed to multiple stresses. This study lays the basis for the proposition of a novel research paradigm which is referred to a holistic approach and that went beyond the exclusive concept of crop yield, but that included sustainability, socio-economic impacts of production, commercialization, and agroecosystem management.

Long-Term Stability in Electronic Properties of Textile Organic Electrochemical Transistors for Integrated Applications.

Organic electrochemical transistors (OECTs) have demonstrated themselves to be an efficient interface between living environments and electronic devices in bioelectronic applications. The peculiar properties of conductive polymers allow new performances that overcome the limits of conventional inorganic biosensors, exploiting the high biocompatibility coupled to the ionic interaction. Moreover, the combination with biocompatible and flexible substrates, such as textile fibers, improves the interaction with living cells and allows specific new applications in the biological environment, including real-time analysis of plants' sap or human sweat monitoring. In these applications, a crucial issue is the lifetime of the sensor device. The durability, long-term stability, and sensitivity of OECTs were studied for two different textile functionalized fiber preparation processes: (i) adding ethylene glycol to the polymer solution, and (ii) using sulfuric acid as a post-treatment. Performance degradation was studied by analyzing the main electronic parameters of a significant number of sensors for a period of 30 days. RGB optical analysis were performed before and after the treatment of the devices. This study shows that device degradation occurs at voltages higher than 0.5 V. The sensors obtained with the sulfuric acid approach exhibit the most stable performances over time.

Field Plant Monitoring from Macro to Micro Scale: Feasibility and Validation of Combined Field Monitoring Approaches from Remote to in Vivo to Cope with Drought Stress in Tomato.

Monitoring plant growth and development during cultivation to optimize resource use efficiency is crucial to achieve an increased sustainability of agriculture systems and ensure food security. In this study, we compared field monitoring approaches from the macro to micro scale with the aim of developing novel in vivo tools for field phenotyping and advancing the efficiency of drought stress detection at the field level. To this end, we tested different methodologies in the monitoring of tomato growth under different water regimes: (i) micro-scale (inserted in the plant stem) real-time monitoring with an organic electrochemical transistor (OECT)-based sensor, namely a bioristor, that enables continuous monitoring of the plant; (ii) medium-scale (<1 m from the canopy) monitoring through red-green-blue (RGB) low-cost imaging; (iii) macro-scale multispectral and thermal monitoring using an unmanned aerial vehicle (UAV). High correlations between aerial and proximal remote sensing were found with chlorophyll-related indices, although at specific time points (NDVI and NDRE with GGA and SPAD). The ion concentration and allocation monitored by the index R of the bioristor during the drought defense response were highly correlated with the water use indices (Crop Water Stress Index (CSWI), relative water content (RWC), vapor pressure deficit (VPD)). A high negative correlation was observed with the CWSI and, in turn, with the RWC. Although proximal remote sensing measurements correlated well with water stress indices, vegetation indices provide information about the crop's status at a specific moment. Meanwhile, the bioristor continuously monitors the ion movements and the correlated water use during plant growth and development, making this tool a promising device for field monitoring.

A Lycopene ε-Cyclase TILLING Allele Enhances Lycopene and Carotenoid Content in Fruit and Improves Drought Stress Tolerance in Tomato Plants.

In the scenario of climate change, the availability of genetic resources for tomato cultivation that combine improved nutritional properties and more tolerance to water deficiency is highly desirable. Within this context, the molecular screenings of the Red Setter cultivar-based TILLING platform led to the isolation of a novel lycopene ε-cyclase gene (SlLCY-E) variant (G/3378/T) that produces modifications in the carotenoid content of tomato leaves and fruits. In leaf tissue, the novel G/3378/T SlLCY-E allele enhances ß,ß-xanthophyll content at the expense of lutein, which decreases, while in ripe tomato fruit the TILLING mutation induces a significant increase in lycopene and total carotenoid content. Under drought stress conditions, the G/3378/T SlLCY-E plants produce more abscisic acid (ABA) and still conserve their leaf carotenoid profile (reduction of lutein and increase in ß,ß-xanthophyll content). Furthermore, under said conditions, the mutant plants grow much better and are more tolerant to drought stress, as revealed by digital-based image analysis and in vivo monitoring of the OECT (Organic Electrochemical Transistor) sensor. Altogether, our data indicate that the novel TILLING SlLCY-E allelic variant is a valuable genetic resource that can be used for developing new tomato varieties, improved in drought stress tolerance and enriched in fruit lycopene and carotenoid content.

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

Increasing the amylose content of durum wheat through silencing of the SBEIIa genes.

BACKGROUND: High amylose starch has attracted particular interest because of its correlation with the amount of Resistant Starch (RS) in food. RS plays a role similar to fibre with beneficial effects for human health, providing protection from several diseases such as colon cancer, diabetes, obesity, osteoporosis and cardiovascular diseases. Amylose content can be modified by a targeted manipulation of the starch biosynthetic pathway. In particular, the inactivation of the enzymes involved in amylopectin synthesis can lead to the increase of amylose content. In this work, genes encoding starch branching enzymes of class II (SBEIIa) were silenced using the RNA interference (RNAi) technique in two cultivars of durum wheat, using two different methods of transformation (biolistic and Agrobacterium). Expression of RNAi transcripts was targeted to the seed endosperm using a tissue-specific promoter. RESULTS: Amylose content was markedly increased in the durum wheat transgenic lines exhibiting SBEIIa gene silencing. Moreover the starch granules in these lines were deformed, possessing an irregular and deflated shape and being smaller than those present in the untransformed controls. Two novel granule bound proteins, identified by SDS-PAGE in SBEIIa RNAi lines, were investigated by mass spectrometry and shown to have strong homologies to the waxy proteins. RVA analysis showed new pasting properties associated with high amylose lines in comparison with untransformed controls. Finally, pleiotropic effects on other starch genes were found by semi-quantitative and Real-Time reverse transcription-polymerase chain reaction (RT-PCR). CONCLUSION: We have found that the silencing of SBEIIa genes in durum wheat causes obvious alterations in granule morphology and starch composition, leading to high amylose wheat. Results obtained with two different methods of transformation and in two durum wheat cultivars were comparable.

Shaping Durum Wheat for the Future: Gene Expression Analyses and Metabolites Profiling Support the Contribution of BCAT Genes to Drought Stress Response.

Global climate change, its implications for agriculture, and the complex scenario presented by the scientific community are of worldwide concern. Drought is a major abiotic stress that can restrict plants growth and yields, thus the identification of genotypes with higher adaptability to drought stress represents one of the primary goals in breeding programs. During abiotic stress, metabolic adaptation is crucial for stress tolerance, and accumulation of specific amino acids and/or as secondary metabolites deriving from amino acid metabolism may correlate with the increased tolerance to adverse environmental conditions. This work, focused on the metabolism of branched chain-amino acids (BCAAs) in durum wheat and the role of branched-chain amino acid aminotransferases (BCATs) in stress response. The role of BCATs in plant response to drought was previously proposed for Arabidopsis, where the levels of BCAAs were altered at the transcriptional level under drought conditions, triggering the onset of defense response metabolism. However, in wheat the role of BCAAs as a trigger of the onset of the drought defense response has not been elucidated. A comparative genomic approach elucidated the composition of the BCAT gene family in durum wheat. Here we demonstrate a tissue and developmental stage specificity of BCATs regulation in the drought response. Moreover, a metabolites profiling was performed on two contrasting durum wheat cultivars Colosseo and Cappelli resulting in the detection of a specific pattern of metabolites accumulated among genotypes and, in particular, in an enhanced BCAAs accumulation in the tolerant cv Cappelli further supporting a role of BCAAs in the drought defense response. The results support the use of gene expression and target metabolomic in modern breeding to shape new cultivars more resilient to a changing climate.

The bean polygalacturonase-inhibiting protein 2 (PvPGIP2) is highly conserved in common bean (Phaseolus vulgaris L.) germplasm and related species.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant protein inhibitors of endo-polygalacturonases (PGs) that belong to the leucine-rich repeat (LRR) protein family. In bean, PGIP is encoded by a small gene family of four members among which Pvpgip2 encodes the most wide-spectrum and efficient inhibitor of fungal PGs. In order to evaluate the sequence polymorphism of Pvpgip2 and its functional significance, we have analyzed a number of wild and cultivated bean (P. vulgaris) accessions of Andean and Mesoamerican origin, and some genotypes from the related species P. coccineus, P. acutifolius, and P. lunatus. Our analyses indicate that the protein encoded by Pvpgip2 is highly conserved in the bean germplasm. The few detected polymorphic sites correspond to synonymous substitutions and only two wild genotypes contain a Pvpgip2 with a single non-synonymous replacement. Sequence comparison showed a slightly larger variation in the related bean species P. coccineus, P. acutifolius, and P. lunatus and confirmed the known phylogenetic relationships with P. vulgaris. The majority of the replacements were within the xxLxLxx region of the leucine rich repeat (LRR) domain and none of them affected residues contributing to structural features. The variant PGIPs were expressed in Nicotiana benthamiana using PVX as vector and their inhibitory activity compared to that of PvPPGIP2. All the variants were able to fully inhibit the four fungal PGs tested with minor differences. Taken together these results support the hypothesis that the overall sequence conservation of PGIP2 and minor variation at specific sites is necessary for high-affinity recognition of different fungal PGs.

Corrigendum: Can High Throughput Phenotyping Help Food Security in the Mediterranean Area?

[This corrects the article DOI: 10.3389/fpls.2019.00015.].

In Vivo Phenotyping for the Early Detection of Drought Stress in Tomato.

Drought stress imposes a major constraint over a crop yield and can be expected to grow in importance if the climate change predicted comes about. Improved methods are needed to facilitate crop management via the prompt detection of the onset of stress. Here, we report the use of an in vivo OECT (organic electrochemical transistor) sensor, termed as bioristor, in the context of the drought response of the tomato plant. The device was integrated within the plant's stem, thereby allowing for the continuous monitoring of the plant's physiological status throughout its life cycle. Bioristor was able to detect changes of ion concentration in the sap upon drought, in particular, those dissolved and transported through the transpiration stream, thus efficiently detecting the occurrence of drought stress immediately after the priming of the defence responses. The bioristor's acquired data were coupled with those obtained in a high-throughput phenotyping platform revealing the extreme complementarity of these methods to investigate the mechanisms triggered by the plant during the drought stress event.