Nitrogen deficiency tolerance conferred by introgression of a QTL derived from wild emmer into bread wheat.

KEY MESSAGE: Genetic dissection of a QTL from wild emmer wheat, QGpc.huj.uh-5B.2, introgressed into bread wheat, identified candidate genes associated with tolerance to nitrogen deficiency, and potentially useful for improving nitrogen-use efficiency. Nitrogen (N) is an important macronutrient critical to wheat growth and development; its deficiency is one of the main factors causing reductions in grain yield and quality. N availability is significantly affected by drought or flooding, that are dependent on additional factors including soil type or duration and severity of stress. In a previous study, we identified a high grain protein content QTL (QGpc.huj.uh-5B.2) derived from the 5B chromosome of wild emmer wheat, that showed a higher proportion of explained variation under water-stress conditions. We hypothesized that this QTL is associated with tolerance to N deficiency as a possible mechanism underlying the higher effect under stress. To validate this hypothesis, we introgressed the QTL into the elite bread wheat var. Ruta, and showed that under N-deficient field conditions the introgression IL99 had a 33% increase in GPC (p < 0.05) compared to the recipient parent. Furthermore, evaluation of IL99 response to severe N deficiency (10% N) for 14 days, applied using a semi-hydroponic system under controlled conditions, confirmed its tolerance to N deficiency. Fine-mapping of the QTL resulted in 26 homozygous near-isogenic lines (BC4F5) segregating to N-deficiency tolerance. The QTL was delimited from - 28.28 to - 1.29 Mb and included 13 candidate genes, most associated with N-stress response, N transport, and abiotic stress responses. These genes may improve N-use efficiency under severely N-deficient environments. Our study demonstrates the importance of WEW as a source of novel candidate genes for sustainable improvement in tolerance to N deficiency in wheat.

Integrative analysis of the methylome and transcriptome of tomato fruit (Solanum lycopersicum L.) induced by postharvest handling.

Tomato fruit ripening is triggered by the demethylation of key genes, which alters their transcriptional levels thereby initiating and propagating a cascade of physiological events. What is unknown is how these processes are altered when fruit are ripened using postharvest practices to extend shelf-life, as these practices often reduce fruit quality. To address this, postharvest handling-induced changes in the fruit DNA methylome and transcriptome, and how they correlate with ripening speed, and ripening indicators such as ethylene, abscisic acid, and carotenoids, were assessed. This study comprehensively connected changes in physiological events with dynamic molecular changes. Ripening fruit that reached 'Turning' (T) after dark storage at 20°C, 12.5°C, or 5°C chilling (followed by 20°C rewarming) were compared to fresh-harvest fruit 'FHT'. Fruit stored at 12.5°C had the biggest epigenetic marks and alterations in gene expression, exceeding changes induced by postharvest chilling. Fruit physiological and chronological age were uncoupled at 12.5°C, as the time-to-ripening was the longest. Fruit ripening to Turning at 12.5°C was not climacteric; there was no respiratory or ethylene burst, rather, fruit were high in abscisic acid. Clear differentiation between postharvest-ripened and 'FHT' was evident in the methylome and transcriptome. Higher expression of photosynthetic genes and chlorophyll levels in 'FHT' fruit pointed to light as influencing the molecular changes in fruit ripening. Finally, correlative analyses of the -omics data putatively identified genes regulated by DNA methylation. Collectively, these data improve our interpretation of how tomato fruit ripening patterns are altered by postharvest practices, and long-term are expected to help improve fruit quality.

CsMLO8/11 are required for full susceptibility of cucumber stem to powdery mildew and interact with CsCRK2 and CsRbohD.

Powdery mildew (PM) is one of the most destructive diseases that threaten cucumber production globally. Efficient breeding of novel PM-resistant cultivars will require a robust understanding of the molecular mechanisms of cucumber resistance against PM. Using a genome-wide association study, we detected a locus significantly correlated with PM resistance in cucumber stem, pm-s5.1. A 1449-bp insertion in the CsMLO8 coding region at the pm-s5.1 locus resulted in enhanced stem PM resistance. Knockout mutants of CsMLO8 and CsMLO11 generated by CRISPR/Cas9 both showed improved PM resistance in the stem, hypocotyl, and leaves, and the double mutant mlo8mlo11 displayed even stronger resistance. We found that reactive oxygen species (ROS) accumulation was higher in the stem of these mutants. Protein interaction assays suggested that CsMLO8 and CsMLO11 could physically interact with CsRbohD and CsCRK2, respectively. Further, we showed that CsMLO8 and CsCRK2 competitively interact with the C-terminus of CsRbohD to affect CsCRK2-CsRbohD module-mediated ROS production during PM defense. These findings provide new insights into the understanding of CsMLO proteins during PM defense responses.

Natural variation in STAYGREEN contributes to low-temperature tolerance in cucumber.

Low-temperature (LT) stress threatens cucumber production globally; however, the molecular mechanisms underlying LT tolerance in cucumber remain largely unknown. Here, using a genome-wide association study (GWAS), we found a naturally occurring single nucleotide polymorphism (SNP) in the STAYGREEN (CsSGR) coding region at the gLTT5.1 locus associated with LT tolerance. Knockout mutants of CsSGR generated by clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 exhibit enhanced LT tolerance, in particularly, increased chlorophyll (Chl) content and reduced reactive oxygen species (ROS) accumulation in response to LT. Moreover, the C-repeat Binding Factor 1 (CsCBF1) transcription factor can directly activate the expression of CsSGR. We demonstrate that the LT-sensitive haplotype CsSGRHapA , but not the LT-tolerant haplotype CsSGRHapG could interact with NON-YELLOW COLORING 1 (CsNYC1) to mediate Chl degradation. Geographic distribution of the CsSGR haplotypes indicated that the CsSGRHapG was selected in cucumber accessions from high latitudes, potentially contributing to LT tolerance during cucumber cold-adaptation in these regions. CsSGR mutants also showed enhanced tolerance to salinity, water deficit, and Pseudoperonospora cubensis, thus CsSGR is an elite target gene for breeding cucumber varieties with broad-spectrum stress tolerance. Collectively, our findings provide new insights into LT tolerance and will ultimately facilitate cucumber molecular breeding.

GWAS reveals novel loci and identifies a pentatricopeptide repeat-containing protein (CsPPR) that improves low temperature germination in cucumber.

Low temperatures (LTs) negatively affect the percentage and rate of cucumber (Cucumis sativus L.) seed germination, which has deleterious effects on yield. Here, a genome-wide association study (GWAS) was used to identify the genetic loci underlying low temperature germination (LTG) in 151 cucumber accessions that represented seven diverse ecotypes. Over two years, phenotypic data for LTG i.e., relative germination rate (RGR), relative germination energy (RGE), relative germination index (RGI) and relative radical length (RRL), were collected in two environments, and 17 of the 151 accessions were found to be highly cold tolerant using cluster analysis. A total of 1,522,847 significantly associated single-nucleotide polymorphism (SNP) were identified, and seven loci associated with LTG, on four chromosomes, were detected: gLTG1.1, gLTG1.2, gLTG1.3, gLTG4.1, gLTG5.1, gLTG5.2, and gLTG6.1 after resequencing of the accessions. Of the seven loci, three, i.e., gLTG1.2, gLTG4.1, and gLTG5.2, showed strong signals that were consistent over two years using the four germination indices, and are thus strong and stable for LTG. Eight candidate genes associated with abiotic stress were identified, and three of them were potentially causal to LTG: CsaV3_1G044080 (a pentatricopeptide repeat-containing protein) for gLTG1.2, CsaV3_4G013480 (a RING-type E3 ubiquitin transferase) for gLTG4.1, and CsaV3_5G029350 (a serine/threonine-protein kinase) for gLTG5.2. The function for CsPPR (CsaV3_1G044080) in regulating LTG was confirmed, as Arabidopsis lines ectopically expressing CsPPR showed higher germination and survival rates at 4°C compared to the wild-type, which preliminarily illustrates that CsPPR positively regulates cucumber cold tolerance at the germination stage. This study will provide insights into cucumber LT-tolerance mechanisms and further promote cucumber breeding development.

Reducing crop losses by gene-editing control of organ developmental physiology.

Some physiological processes in reproductive organs, if not controlled, can lead to crop loss even in the absence of environmental stress. These processes may occur pre- or post- harvest, and in diverse species and include abscission processes in cereal grain, e.g., shattering and in immature fruit, e.g., preharvest drop, preharvest sprouting of cereals, and postharvest senescence in fruit. Some of the molecular mechanisms and genetic determinants underlying these processes are now better detailed, making it possible to refine them by gene editing. Here, we discuss using advanced genomics to identify genetic determinants underlying crop physiological traits. Examples of improved phenotypes developed for preharvest problems are provided, and suggestions for reducing postharvest fruit losses by gene and promoter editing were made.

Starch and sugars as determinants of postharvest shelf life and quality: some new and surprising roles.

Starch and sugars account for most of the dry weight of horticultural crops and in many species, are known determinants of quality. However, we posit that these carbohydrates often have less-obvious roles in plant tissues with direct implications for the postharvest quality and produce shelf life. The latter has not been given as much attention, but with the recent interest in reducing the scale of postharvest waste and loss, we highlight how dynamic changes in the spatial-temporal accumulation of carbohydrates, can influence myriads of biological processes affecting postharvest attributes. Versatile roles, some surprising, that carbohydrates play in determining produce of high value to consumers, are highlighted, and gene targets for biotechnological improvement are specified.

Ectopic Expression of Arabidopsis thaliana zDof1.3 in Tomato (Solanum lycopersicum L.) Is Associated with Improved Greenhouse Productivity and Enhanced Carbon and Nitrogen Use.

A large collection of transgenic tomato lines, each ectopically expressing a different Arabidopsis thaliana transcription factor, was screened for variants with alterations in leaf starch. Such lines may be affected in carbon partitioning, and in allocation to the sinks. We focused on 'L4080', which harbored an A. thaliana zDof (DNA-binding one zinc finger) isoform 1.3 (AtzDof1.3) gene, and which had a 2−4-fold higher starch-to-sucrose ratio in source leaves over the diel (p < 0.05). Our aim was to determine whether there were associated effects on productivity. L4080 plants were altered in nitrogen (N) and carbon (C) metabolism. The N-to-C ratio was higher in six-week-old L4080, and when treated with 1/10 N, L4080 growth was less inhibited compared to the wild-type and this was accompanied by faster root elongation (p < 0.05). The six-week-old L4080 acquired 42% more dry matter at 720 ppm CO2, compared to ambient CO2 (p < 0.05), while the wild-type (WT) remained unchanged. GC-MS-TOF data showed that L4080 source leaves were enriched in amino acids compared to the WT, and at 49 DPA, fruit had 25% greater mass, higher sucrose, and increased yield (25%; p < 0.05) compared to the WT. An Affymetrix cDNA array analysis suggested that only 0.39% of the 9000 cDNAs were altered by 1.5-fold (p < 0.01) in L4080 source leaves. 14C-labeling of fruit disks identified potential differences in 14-DPA fruit metabolism suggesting that post-transcriptional regulation was important. We conclude that AtzDof1.3 and the germplasm derived therefrom, should be investigated for their 'climate-change adaptive' potential.

Physiological and Metabolic Responses of Gac Leaf (Momordica cochinchinensis (Lour.) Spreng.) to Salinity Stress.

Gac is a carotenoid-rich, healthful tropical fruit; however, its productivity is limited by soil salinity, a growing environmental stress. This study aimed to evaluate the effects of salinity stress on key physiological traits and metabolites in 30-day-old gac seedling leaves, treated with 0, 25-, 50-, 100-, and 150-mM sodium chloride (NaCl) for four weeks to identify potential alarm, acclimatory, and exhaustion responses. Electrolyte leakage increased with increasing NaCl concentrations (p < 0.05) indicating loss of membrane permeability and conditions that lead to reactive oxygen species production. At 25 and 50 mM NaCl, superoxide dismutase (SOD) activity, starch content, and total soluble sugar increased. Chlorophyll a, and total chlorophyll increased at 25 mM NaCl but decreased at higher NaCl concentrations indicating salinity-induced thylakoid membrane degradation and chlorophyllase activity. Catalase (CAT) activity decreased (p < 0.05) at all NaCl treatments, while ascorbate peroxidase (APX) and guaiacol peroxidase (GPX) activities were highest at 150 mM NaCl. GC-MS-metabolite profiling showed that 150 mM NaCl induced the largest changes in metabolites and was thus distinct. Thirteen pathways and 7.73% of metabolites differed between the control and all the salt-treated seedlings. Salinity decreased TCA cycle intermediates, and there were less sugars for growth but more for osmoprotection, with the latter augmented by increased amino acids. Although 150 mM NaCl level decreased SOD activity, the APX and GPX enzymes were still active, and some carbohydrates and metabolites also accumulated to promote salinity resistance via multiple mechanisms.

Dissecting postharvest chilling injury through biotechnology.

Paradoxically, refrigerating many fruits and vegetables destroys their quality, and may even accelerate their spoilage. This phenomenon, known as postharvest chilling injury (PCI), affects produce from tropical and subtropical regions and leads to economic and postharvest loss and waste. Low temperatures are used to pause the physiological processes associated with senescence, but upon rewarming, these processes may resume at an accelerated rate. Chilling-injured produce may be discarded for not meeting consumer expectations or may prematurely deteriorate. In this review, we describe progress made in identifying the cellular and molecular processes underlying PCI, and point to advances in biotechnological approaches for ameliorating symptoms. Further, we identify the gaps in knowledge that must be bridged to develop effective solutions to PCI.

Fine mapping a quantitative trait locus underlying seedling resistance to gummy stem blight using a residual heterozygous lines-derived strategy in cucumber.

Gummy stem blight (GSB), caused by Didymella bryoniae, is one of the most devastating diseases that severely reduces cucumber production. Developing resistant varieties would be an effective strategy to control GSB. Although several GSB-resistant QTLs have been reported, causal genes for GSB resistance have not yet been identified in cucumber. A novel loci gsb3.1 for seedling GSB resistance from the "PI 183967" genotype was previously identified in a 1.7-Mb interval on chromosome 3. In this study, we developed a residual heterozygous line-derived strategy from Recombinant Inbred Lines to perform fine mapping, and with this approach, the gsb3.1 locus was narrowed to a 38 kb interval. There were six predicted genes at the gsb3.1 locus, four of which differed in expression in the GSB-resistant compared to the susceptible lines after fungal inoculation. These candidate genes (Csa3G020050, Csa3G020060, Csa3G020090, and Csa3G020590) within the gsb3.1 locus could be helpful for the genetic study of GSB resistance and marker-assisted selection in cucumber. Phylogenetic analyses indicated that the resistant gsb3.1 allele may uniquely exist in the wild species present in the Indian group, and that nucleotide diversity was significantly reduced in cultivated accessions. Therefore, the gsb3.1 allele could be introgressed into existing commercial cultivars and combined with other resistance QTLs to provide broad-spectrum and robust GSB resistance in cucumber.

Genome-Wide Association Study in Bread Wheat Identifies Genomic Regions Associated with Grain Yield and Quality under Contrasting Water Availability.

The objectives of this study were to identify genetic loci in the bread wheat genome that would influence yield stability and quality under water stress, and to identify accessions that can be recommended for cultivation in dry and hot regions. We performed a genome-wide association study (GWAS) using a panel of 232 wheat accessions spanning diverse ecogeographic regions. Plants were evaluated in the Israeli Northern Negev, under two environments: water-limited (D; 250 mm) and well-watered (W; 450 mm) conditions; they were genotyped with ~71,500 SNPs derived from exome capture sequencing. Of the 14 phenotypic traits evaluated, 12 had significantly lower values under D compared to W conditions, while the values for two traits were higher under D. High heritability (H2 = 0.5-0.9) was observed for grain yield, spike weight, number of grains per spike, peduncle length, and plant height. Days to heading and grain yield could be partitioned based on accession origins. GWAS identified 154 marker-trait associations (MTAs) for yield and quality-related traits, 82 under D and 72 under W, and identified potential candidate genes. We identified 24 accessions showing high and/or stable yields under D conditions that can be recommended for cultivation in regions under the threat of global climate change.

Fine mapping and candidate gene analysis of gummy stem blight resistance in cucumber stem.

KEY MESSAGE: Two candidate genes (Csa6G046210 and Csa6G046240) were identified by fine-mapping gsb-s6.2 for gummy stem blight resistance in cucumber stem. Gummy stem blight (GSB) is a serious fungal disease caused by Didymella bryoniae, that affects cucumber yield and quality worldwide. However, no GSB-resistant genes have been identified in cucumber cultivars. In this study, the wild cucumber accession 'PI 183967' was used as a source of resistance to GSB in adult stems. An F2 population was mapped using resistant line 'LM189' and susceptible line 'LM6' derived from a cross between 'PI 183967' and '931'. By developing InDel and SNP markers, the gsb-s6.2 QTL on Chr. 6 was fine-mapped to a 34 kb interval harboring six genes. Gene Expression analysis after inoculation showed that two candidate genes (Csa6G046210 and Csa6G046240) were induced and differentially expressed between the resistant and susceptible parents, and may be involved in disease defense. Sequence alignment showed that Csa6G046210 encodes a multiple myeloma tumor-associated protein, and it harbored two nonsynonymous SNPs and one InDel in the third and the fourth exons, and two InDels in the TATA-box of the basal promoter region. Csa6G046240 encodes a MYB transcription factor with six variants in the AP2/ERF and MYB motifs in the promoter. These two candidate genes lay the foundation for revealing the mechanism of GSB resistance and may be useful for marker-assisted selection in cucumber disease-resistant breeding.

The qLTG1.1 candidate gene CsGAI regulates low temperature seed germination in cucumber.

KEY MESSAGE: The CsGAI gene, identified by map-based, was involved in regulating seed germination in low temperature via the GA and ABA signaling pathways. Low temperature reduces the percentage of seeds germinating and delays seed germinating time, thus posing a threat to cucumber production. However, the molecular mechanism regulating low temperature germination in cucumber is unknown. We here dissected a major quantitative trait locus qLTG1.1 that controls seed germination at low temperature in cucumber. First, we fine-mapped qLTG1.1 to a 46.3-kb interval, containing three candidate genes. Sequence alignment and gene expression analysis identified Csa1G408720 as the gene of interest that was highly expressed in seeds, and encoded a highly conserved, low temperature-regulated DELLA family protein CsGAI. GUS expression analysis indicated that higher promoter activity underscored higher transcriptional expression of the CsGAI gene. Consistent with the known roles of GAI in ABA and GA signaling during germination, genes involved in the GA (CsGA2ox, CsGA3ox) and ABA biosynthetic pathways (CsABA1, CsABA2, CsAAO3 and CsNCED) were found to be differently regulated in the tolerant and sensitive genotypes under low temperatures, and this was reflected in differences in their ratio of GA-to-ABA. Based on these data, we proposed a working model explaining how CsGAI integrates the GA and ABA signaling pathways, to regulate cucumber seed germination at low temperature, thus providing new insights into this mechanism.

Starch branching enzymes as putative determinants of postharvest quality in horticultural crops.

Starch branching enzymes (SBEs) are key determinants of the structure and amount of the starch in plant organs, and as such, they have the capacity to influence plant growth, developmental, and fitness processes, and in addition, the industrial end-use of starch. However, little is known about the role of SBEs in determining starch structure-function relations in economically important horticultural crops such as fruit and leafy greens, many of which accumulate starch transiently. Further, a full understanding of the biological function of these types of starches is lacking. Because of this gap in knowledge, this minireview aims to provide an overview of SBEs in horticultural crops, to investigate the potential role of starch in determining postharvest quality. A systematic examination of SBE sequences in 43 diverse horticultural species, identified SBE1, 2 and 3 isoforms in all species examined except apple, olive, and Brassicaceae, which lacked SBE1, but had a duplicated SBE2. Among our findings after a comprehensive and critical review of published data, was that as apple, banana, and tomato fruits ripens, the ratio of the highly digestible amylopectin component of starch increases relative to the more digestion-resistant amylose fraction, with parallel increases in SBE2 transcription, fruit sugar content, and decreases in starch. It is tempting to speculate that during the ripening of these fruit when starch degradation occurs, there are rearrangements made to the structure of starch possibly via branching enzymes to increase starch digestibility to sugars. We propose that based on the known action of SBEs, and these observations, SBEs may affect produce quality, and shelf-life directly through starch accumulation, and indirectly, by altering sugar availability. Further studies where SBE activity is fine-tuned in these crops, can enrich our understanding of the role of starch across species and may improve horticulture postharvest quality.

Crops for Carbon Farming.

Agricultural cropping systems and pasture comprise one third of the world's arable land and have the potential to draw down a considerable amount of atmospheric CO2 for storage as soil organic carbon (SOC) and improving the soil carbon budget. An improved soil carbon budget serves the dual purpose of promoting soil health, which supports crop productivity, and constituting a pool from which carbon can be converted to recalcitrant forms for long-term storage as a mitigation measure for global warming. In this perspective, we propose the design of crop ideotypes with the dual functionality of being highly productive for the purposes of food, feed, and fuel, while at the same time being able to facilitate higher contribution to soil carbon and improve the below ground ecology. We advocate a holistic approach of the integrated plant-microbe-soil system and suggest that significant improvements in soil carbon storage can be achieved by a three-pronged approach: (1) design plants with an increased root strength to further allocation of carbon belowground; (2) balance the increase in belowground carbon allocation with increased source strength for enhanced photosynthesis and biomass accumulation; and (3) design soil microbial consortia for increased rhizosphere sink strength and plant growth-promoting (PGP) properties.

Lipidomic and metabolomic profiles of Coffea canephora L. beans cultivated in Southwestern Nigeria.

Coffee (Coffea spp.) is one of the most popular refreshing beverages globally. Coffee lipid diversity has untapped potential for improving coffee marketability because lipids contribute significantly to both the health benefits and cup quality of coffee. However, in spite of its potential importance, there have not been extensive studies of lipids among C. canephora genotypes. In this study, ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) profiling of lipid molecules was performed for 30 genotypes consisting of 15 cultivated and 15 conserved genotypes of C. canephora in Southwestern Nigeria. We identified nine classes of lipids in the 30 genotypes which belong to the 'Niaouli', 'Kouillou' and 'Java Robusta' group: among these, the most abundant lipid class was the triacylglycerols, followed by the fatty acyls group. Although 'Niaouli' diverged from the 'Kouillou' and 'Java Robusta' genotypes when their lipid profiles were compared, there was greater similarity in their lipid composition by multivariate analysis, compared to that observed when their primary metabolites and especially their secondary metabolite profiles were examined. However, distinctions could be made among genotypes. Members of the fatty acyls group had the greatest power to discriminate among genotypes, however, lipids that were low in abundance e.g. a cholesterol ester (20:3), and phosphotidylethanolamine (34:0) were also helpful to understand the relationships among C. canephora genotypes. The divergent lipid profiles identified among the C. canephora genotypes, correlated with their Single Nucleotide Polymorphism grouping as assessed by genotype-by-sequencing, and will be exploited to improve coffee cup quality.