Cross-species transcriptomic analyses reveals common and opposite responses in Arabidopsis, rice and barley following oxidative stress and hormone treatment.

BACKGROUND: For translational genomics, a roadmap is needed to know the molecular similarities or differences between species, such as model species and crop species. This knowledge is invaluable for the selection of target genes and pathways to alter downstream in response to the same stimuli. Here, the transcriptomic responses to six treatments including hormones (abscisic acid - ABA and salicylic acid - SA); treatments that cause oxidative stress (3-amino-1,2,4-triazole - 3AT, methyl viologen - MV); inhibit respiration (antimycin A - AA) or induce genetic damage (ultraviolet radiation -UV) were analysed and compared between Arabidopsis (Arabidopsis thaliana), barley (Hordeum vulgare) and rice (Oryza sativa). RESULTS: Common and opposite responses were identified between species, with the number of differentially expressed genes (DEGs) varying greatly between treatments and species. At least 70% of DEGs overlapped with at least one other treatment within a species, indicating overlapping response networks. Remarkably, 15 to 34% of orthologous DEGs showed opposite responses between species, indicating diversity in responses, despite orthology. Orthologous DEGs with common responses to multiple treatments across the three species were correlated with experimental data showing the functional importance of these genes in biotic/abiotic stress responses. The mitochondrial dysfunction response was revealed to be highly conserved in all three species in terms of responsive genes and regulation via the mitochondrial dysfunction element. CONCLUSIONS: The orthologous DEGs that showed a common response between species indicate conserved transcriptomic responses of these pathways between species. However, many genes, including prominent salt-stress responsive genes, were oppositely responsive in multiple-stresses, highlighting fundamental differences in the responses and regulation of these genes between species. This work provides a resource for translation of knowledge or functions between species.

Harnessing root architecture to address global challenges.

Root architecture can be targeted in breeding programs to develop crops with better capture of water and nutrients. In rich nations, such crops would reduce production costs and environmental pollution and, in developing nations, they would improve food security and economic development. Crops with deeper roots would have better climate resilience while also sequestering atmospheric CO2 . Deeper rooting, which improves water and N capture, is facilitated by steeper root growth angles, fewer axial roots, reduced lateral branching, and anatomical phenotypes that reduce the metabolic cost of root tissue. Mechanical impedance, hypoxia, and Al toxicity are constraints to subsoil exploration. To improve topsoil foraging for P, K, and other shallow resources, shallower root growth angles, more axial roots, and greater lateral branching are beneficial, as are metabolically cheap roots. In high-input systems, parsimonious root phenotypes that focus on water capture may be advantageous. The growing prevalence of Conservation Agriculture is shifting the mechanical impedance characteristics of cultivated soils in ways that may favor plastic root phenotypes capable of exploiting low resistance pathways to the subsoil. Root ideotypes for many low-input systems would not be optimized for any one function, but would be resilient against an array of biotic and abiotic challenges. Root hairs, reduced metabolic cost, and developmental regulation of plasticity may be useful in all environments. The fitness landscape of integrated root phenotypes is large and complex, and hence will benefit from in silico tools. Understanding and harnessing root architecture for crop improvement is a transdisciplinary opportunity to address global challenges.

Exploration of physiological and biochemical processes of canola with exogenously applied fertilizers and plant growth regulators under drought stress.

Environmental stresses may alter the nutritional profile and economic value of crops. Chemical fertilizers and phytohormones are major sources which can enhance the canola production under stressful conditions. Physio-biochemical responses of canola altered remarkably with the use of nitrogen/phosphorus/potassium (N/P/K) fertilizers and plant growth regulators (PGRs) under drought stress. The major aim of current study was to evaluate nutritional quality and physio-biochemical modulation in canola (Brassica napus L.) from early growth to seed stage with NPK and PGRs in different water regimes. To monitor biochemical and physiological processes in canola, two season field experiment was conducted as spilt plot under randomized complete block design (RCBD) with four treatments (Control, Chemical fertilizers [N (90 kg/ha), P and K (45 kg ha-1)], PGRs; indole acetic acid (IAA) 15g ha-1, gibberellic acid (GA3) 15g ha-1 and the combination of NPK and PGRs] under different irrigations regimes (60, 100, 120, 150 mm evaporations). Water stress enhanced peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), polyphenol oxidase (PPO), soluble sugar, malondialdehyde (MDA), proline contents as well as leaf temperature while substantially reduced leaf water contents (21%), stomatal conductance (50%), chlorophyll contents (10-67%), membrane stability index (24%) and grain yield (30%) of canola. However, the combined application of NPK and PGR further increased the enzymatic antioxidant pool, soluble sugars, along with recovery of leaf water contents, chlorophyll contents, stomatal conductance and membrane stability index but decreased the proline contents and leaf temperature at different rate of evaporation. There is positive interaction of applied elicitors to the water stress in canola except leaf area. The outcomes depicted that the combination of NPK with PGRs improved the various morpho-physiological as well as biochemical parameters and reduced the pressure of chemical fertilizers cost about 60%. It had also reduced the deleterious effect of water limitation on the physiology and grain yield and oil contents of canola in field experiments.

Genome-wide analysis of ZAT gene family revealed GhZAT6 regulates salt stress tolerance in G. hirsutum.

High salt environments can induce stress in different plants. The genes containing the ZAT domain constitute a family that belongs to a branch of the C2H2 family, which plays a vital role in responding to abiotic stresses. In this study, we identified 169 ZAT genes from seven plant species, including 44 ZAT genes from G. hirsutum. Phylogenetic tree analysis divided ZAT genes in six groups with conserved gene structure, protein motifs. Two C2H2 domains and an EAR domain and even chromosomal distribution on At and Dt sub-genome chromosomes of G. hirsutum was observed. GhZAT6 was primarily expressed in the root tissue and responded to NaCl and ABA treatments. Subcellular localization found that GhZAT6 was located in the nucleus and demonstrated transactivation activity during a transactivation activity assay. Arabidopsis transgenic lines overexpressing the GhZAT6 gene showed salt tolerance and grew more vigorously than WT on MS medium supplemented with 100 mmol NaCl. Additionally, the silencing of the GhZAT6 gene in cotton plants showed more obvious leaf wilting than the control plants, which were subjected to 400 mmol NaCl treatment. Next, the expressions of GhAPX1, GhFSD1, GhFSD2, and GhSOS3 were significantly lower in the GhZAT6-silenced plants treated with NaCl than the control. Based on these findings, GhZAT6 may be involved in the ABA pathway and mediate salt stress tolerance by regulating ROS-related gene expression.

Identification of six CPC-like genes and their differential expression in leaves of tea plant, Camellia sinensis.

Tea is one of the most consumed beverages worldwide, and trichome formation in tea plant leaves impairs their commercial value. In Arabidopsis thaliana leaves, trichome formation is negatively regulated by the CPC family genes, which encode R3-type MYB transcription factors. Here, we identified six CPC-like genes in a tea plant (Camellia sinensis var. sinensis) for the first time. Simulated three-dimensional structure of the MYB domains of all the six CPC-like proteins exhibited negative charge on the surface, as observed on that of the Arabidopsis CPC protein that does not bind to DNA, indicating their similarity with regard to molecular interaction. We further found that the six CPC-like genes were differentially expressed in different developmental stages of tea leaves, and four out of the six genes were upregulated in the youngest 1st leaves, which formed more trichomes than other older leaves. Although it does not establish a causal link, the correlation between differential expression of CPC-like genes and variable trichome formation suggests that the R3-type MYB transcription factors are potential precipitating factors in affecting the value of tea leaf.

Haploid and Doubled Haploid Plant Production in Brassica rapa L. subsp. pekinensis Via Microspore Culture.

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.

Protocol of European Radish (Raphanus sativus L.) Microspore Culture for Doubled Haploid Plant Production.

Here, we describe the first protocol of European radish (Raphanus sativus L. subsp. sativus convar. radicula) for obtaining doubled haploid plants through in vitro microspore culture, in which the full cycle of doubled haploid formation was successfully achieved. Using this protocol, a yield of up to eight embryoids per Petri dish can be obtained. Effectiveness of this protocol was confirmed for several genotypes of European radish.

Anther and Isolated Microspore Culture in Eggplant (Solanum melongena L.).

Eggplant is one of the five important, worldwide-distributed solanaceous crops. The use of anther culture technology to produce pure, 100% homozygous doubled haploid lines for hybrid seed production is possible since 1982, where the first protocol of wide application to different eggplant materials was published. From then on, different improvements and adaptations to different materials have been made. In parallel, protocols to implement isolated microspore culture technology in eggplant have been developed principally in the last decade, which opens the door for a more efficient DH production in this species. In this chapter, two protocols, one for anther and other for isolated microspore culture in eggplant, are described. Some steps and materials are common to both approaches. A detailed description of each step from is provided.

Shed-Microspore Culture in Ornamental Peppers for Doubled Haploid Plant Production.

The shed-microspore culture technique is an alternative sub-method combining anther and isolated microspore culture to induce microspore embryogenesis. Recently, its effective use in different types of peppers has drawn attention, because it has a higher embryo yield potential compared to anther culture and is more practical than isolated microspore culture. In this chapter, a stepwise protocol for shed-microspore culture of ornamental pepper is described. This protocol includes the steps of donor plant growth conditions, the choice of suitable flower buds based on DAPI staining of microspores, application of a cold pretreatment to flower buds, surface sterilization of the buds, shed-microspore culture of anthers, stress treatments, regeneration of androgenic in vitro plantlets, their acclimatization and ploidy analysis, and in vivo chromosome doubling of the haploid plants.

Zinc oxide nanoparticles alleviates the adverse effects of cadmium stress on Oryza sativa via modulation of the photosynthesis and antioxidant defense system.

Cadmium (Cd) is a trace element causing severe toxicity symptoms in plants, besides posing hazardous fitness issue due to its buildup in the human body through food chain. Nanoparticles (NPs) are recently employed as a novel strategy to directly ameliorate the Cd stress and acted as nano-fertilizers. The intend of the current study was to explore the effects of zinc oxide nanoparticles (ZnO-NPs; 50 mg/L) on plant growth, photosynthetic activity, elemental status and antioxidant activity in Oryza sativa (rice) under Cd (0.8 mM) stress. To this end, the rice plants are treated by Cd stress at 15 days after sowing (DAS), and the treatment was given directly into the soil. Supply of ZnO-NPs as foliar spray was given for five consecutive days from 30 to 35 DAS, and sampling was done at 45 DAS. However, rice plants supplemented with ZnO-NPs under the Cd toxicity revealed significantly increased shoot length (SL; 34.0%), root fresh weight (RFW; 30.0%), shoot dry weight (SDW; 23.07%), and root dry weight (RDW; 12.24%). Moreover, the ZnO-NPs supplement has also positive effects on photosynthesis related parameters, SPAD value (40%), chloroplast structure, and qualitatively high fluorescence observed by confocal microscopy even under Cd stress. ZnO-NPs also substantially prevented the increases of hydrogen peroxide (H2O2) and malondialdehyde (MDA) triggered by Cd. Physiological and biochemical analysis showed that ZnO-NPs increased enzymatic activities of superoxide dismutase (SOD; 59%), catalase (CAT; 52%), and proline (17%) that metabolize reactive oxygen species (ROS); these increases coincided with the changes observed in the H2O2 and MDA accumulation after ZnO-NPs application. In conclusion, ZnO-NPs application to foliage has great efficiency to improve biomass, photosynthesis, protein, antioxidant enzymes activity, mineral nutrient contents and reducing Cd levels in rice. This can be attributed mainly from reduced oxidative damage resulted due to the ZnO-NPs application.

Arabica-like flavour in a heat-tolerant wild coffee species.

There are numerous factors to consider when developing climate-resilient coffee crops, including the ability to tolerate altered climatic conditions, meet agronomic and value chain criteria, and satisfy consumer preferences for flavour (aroma and taste). We evaluated the sensory characteristics and key environmental requirements for the enigmatic narrow-leaved coffee (Coffea stenophylla), a wild species from Upper West Africa1. We confirm historical reports of a superior flavour1-3 and uniquely, and remarkably, reveal a sensory profile analogous to high-quality Arabica coffee. We demonstrate that this species grows and crops under the same range of key climatic conditions as (sensorially inferior) robusta and Liberica coffee4-9 and at a mean annual temperature 6.2-6.8 °C higher than Arabica coffee, even under equivalent rainfall conditions. This species substantially broadens the climate envelope for high-quality coffee and could provide an important resource for the development of climate-resilient coffee crop plants.

A multiple ion-uptake phenotyping platform reveals shared mechanisms affecting nutrient uptake by roots.

Nutrient uptake is critical for crop growth and is determined by root foraging in soil. Growth and branching of roots lead to effective root placement to acquire nutrients, but relatively little is known about absorption of nutrients at the root surface from the soil solution. This knowledge gap could be alleviated by understanding sources of genetic variation for short-term nutrient uptake on a root length basis. A modular platform called RhizoFlux was developed for high-throughput phenotyping of multiple ion-uptake rates in maize (Zea mays L.). Using this system, uptake rates were characterized for the crop macronutrients nitrate, ammonium, potassium, phosphate, and sulfate among the Nested Association Mapping (NAM) population founder lines. The data revealed substantial genetic variation for multiple ion-uptake rates in maize. Interestingly, specific nutrient uptake rates (nutrient uptake rate per length of root) were found to be both heritable and distinct from total uptake and plant size. The specific uptake rates of each nutrient were positively correlated with one another and with specific root respiration (root respiration rate per length of root), indicating that uptake is governed by shared mechanisms. We selected maize lines with high and low specific uptake rates and performed an RNA-seq analysis, which identified key regulatory components involved in nutrient uptake. The high-throughput multiple ion-uptake kinetics pipeline will help further our understanding of nutrient uptake, parameterize holistic plant models, and identify breeding targets for crops with more efficient nutrient acquisition.

Selenium Alleviates the Adverse Effect of Drought in Oilseed Crops Camelina (Camelina sativa L.) and Canola (Brassica napus L.).

Drought poses a serious threat to oilseed crops by lowering yield and crop failures under prolonged spells. A multi-year field investigation was conducted to enhance the drought tolerance in four genotypes of Camelina and canola by selenium (Se) application. The principal aim of the research was to optimize the crop yield by eliciting the physio-biochemical attributes by alleviating the adverse effects of drought stress. Both crops were cultivated under control (normal irrigation) and drought stress (skipping irrigation at stages i.e., vegetative and reproductive) conditions. Four different treatments of Se viz., seed priming with Se (75 µM), foliar application of Se (7.06 µM), foliar application of Se + Seed priming with Se (7.06 µM and 75 µM, respectively) and control (without Se), were implemented at the vegetative and reproductive stages of both crops. Sodium selenite (Na2SeO3), an inorganic compound was used as Se sources for both seed priming and foliar application. Data regarding physiochemical, antioxidants, and yield components were recorded as response variables at crop maturity. Results indicated that WP, OP, TP, proline, TSS, TFAA, TPr, TS, total chlorophyll contents, osmoprotectant (GB, anthocyanin, TPC, and flavonoids), antioxidants (APX, SOD, POD, and CAT), and yield components (number of branches per plant, thousand seed weight, seed, and biological yields were significantly improved by foliar Se + priming Se in both crops under drought stress. Moreover, this treatment was also helpful in boosting yield attributes under irrigated (non-stress) conditions. Camelina genotypes responded better to Se application as seed priming and foliar spray than canola for both years. It has concluded that Se application (either foliar or priming) can potentially alleviate adverse effects of drought stress in camelina and canola by eliciting various physio-biochemicals attributes under drought stress. Furthermore, Se application was also helpful for crop health under irrigated condition.

Root hydraulics adjustment is governed by a dominant cell-to-cell pathway in Beta vulgaris seedlings exposed to salt stress.

Soil salinity reduces root hydraulic conductivity (Lpr) of several plant species. However, how cellular signaling and root hydraulic properties are linked in plants that can cope with water restriction remains unclear. In this work, we exposed the halotolerant species red beet (Beta vulgaris) to increasing concentrations of NaCl to determine the components that might be critical to sustaining the capacity to adjust root hydraulics. Our strategy was to use both hydraulic and cellular approaches in hydroponically grown seedlings during the first osmotic phase of salt stress. Interestingly, Lpr presented a bimodal profile response apart from the magnitude of the imposed salt stress. As well as Lpr, the PIP2-aquaporin profile follows an unphosphorylated/phosphorylated pattern when increasing NaCl concentration while PIP1 aquaporins remain constant. Lpr also shows high sensitivity to cycloheximide. In low NaCl concentrations, Lpr was high and 70 % of its capacity could be attributed to the CHX-inhibited cell-to-cell pathway. More interestingly, roots can maintain a constant spontaneous exudated flow that is independent of the applied NaCl concentration. In conclusion, Beta vulgaris root hydraulic adjustment completely lies in a dominant cell-to-cell pathway that contributes to satisfying plant water demands.

Development of broad-spectrum and sustainable resistance in cotton against major insects through the combination of Bt and plant lectin genes.

KEY MESSAGE: Second generation Bt insecticidal toxin in comibination with Allium sativum leaf agglutinin gene has been successfully expressed in cotton to develop sustainable resistance against major chewing and sucking insects. The first evidence of using the Second-generation Bt gene in combination with Allium sativum plant lectin to develop sustainable resistance against chewing and sucking insects has been successfully addressed in the current study. Excessive use of Bt δ-endotoxins in the field is delimiting its insecticidal potential. Second-generation Bt Vip3Aa could be the possible alternative because it does not share midgut receptor sites with any known cry proteins. Insecticidal potential of plant lectins against whitefly remains to be evaluated. In this study, codon-optimized synthetic Bt Vip3Aa gene under CaMV35S promoter and Allium sativum leaf agglutinin gene under phloem-specific promoter were transformed in a local cotton variety. Initial screening of putative transgenic cotton plants was done through amplification, histochemical staining and immunostrip assay. The mRNA expression of Vip3Aa gene was increased to be ninefold in transgenic cotton line L6P3 than non-transgenic control while ASAL expression was found to be fivefold higher in transgenic line L34P2 as compared to non-transgenic control. The maximum Vip3Aa concentration was observed in transgenic line L6P3. Two copy numbers in homozygous form at chromosome number 9 and one copy number in hemizygous form at chromosome number 10 was observed in transgenic line L6P3 through fluorescent in situ hybridization. Significant variation was observed in transgenic cotton lines for morphological characteristics, whereas physiological parameters of plants and fiber characteristics (as assessed by scanning electron microscopic) remained comparable in transgenic and non-transgenic cotton lines. Leaf-detach bioassay showed that all the transgenic lines were significantly resistant to Helicoverpa armigera showing mortality rates between 78% and 100%. Similarly, up to 95% mortality of whiteflies was observed in transgenic cotton lines when compared with non-transgenic control lines.

Plant resilience to phosphate limitation: current knowledge and future challenges.

Phosphorus (P) is an essential macronutrient for all living organisms. Importantly, plants require a large amount of P to grow, and P deficiency causes huge losses in plant production. Although this issue can be mitigated by the appropriate use of phosphate (Pi) rock-derived P fertilizers, phosphate rock is a finite natural resource. Moreover, the increased demand for food as a result of our growing global population is another factor contributing to a prospective P crisis. While creating crops that are resilient to Pi deficiency presents great scientific challenge, the current progress in our understanding of how plants regulate Pi homeostasis offers some opportunities for further study. In this review, we present the published research supporting these opportunities, which are based on the molecular mechanisms that plants have evolved to respond to P deficiency. First, we focus on recent advances in P sensing and signaling pathways in the regulation of root system architecture. Next, we describe the mechanisms that regulate Pi transport and accumulation, in a Pi- (or other nutrient) dependent manner. Integrating these data will help to design an innovative strategy for improving Pi nutrition in plants. In addition, this will help with Pi scarcity, one of the challenges facing agriculture in the twenty first century.

Marker assisted improvement of low soil phosphorus tolerance in the bacterial blight resistant, fine-grain type rice variety, Improved Samba Mahsuri.

Improved-Samba-Mahsuri (ISM), a high-yielding, popular bacterial blight resistant (possessing Xa21, xa13, and xa5), fine-grain type, low glycemic index rice variety is highly sensitive to low soil phosphorus (P). We have deployed marker-assisted backcross breeding (MABB) approach for targeted transfer of Pup1, a major QTL associated with low soil P tolerance, using Swarna as a donor. A new co-dominant marker, K20-1-1, which is specific for Pup1 was designed and used for foreground selection along with functional markers specific for the bacterial blight resistance genes, Xa21, xa13, and xa5. A set of 66 polymorphic SSR marker were used for the background selection along with a pair of flanking markers for the recombination selection in backcross derived progenies and in BC2F2 generation, 12 plants, which are homozygous for Pup1, all the three bacterial blight resistance genes and possessing agro-morphological traits equivalent to or better than ISM were selected and selfed to produce BC2F3s. They were evaluated in plots with low soil P and normal soil P at ICAR-IIRR, Hyderabad for their low soil P tolerance, and bacterial blight resistance and superior lines were advanced to BC2F6. One of the lines, when tested at multiple locations in India was found promising under both normal as well as low soil P conditions.

The Use of Genetic and Gene Technologies in Shaping Modern Rapeseed Cultivars (Brassica napus L.).

Since their domestication, Brassica oilseed species have undergone progressive transformation allied with the development of breeding and molecular technologies. The canola (Brassica napus) crop has rapidly expanded globally in the last 30 years with intensive innovations in canola varieties, providing for a wider range of markets apart from the food industry. The breeding efforts of B. napus, the main source of canola oil and canola meal, have been mainly focused on improving seed yield, oil quality, and meal quality along with disease resistance, abiotic stress tolerance, and herbicide resistance. The revolution in genetics and gene technologies, including genetic mapping, molecular markers, genomic tools, and gene technology, especially gene editing tools, has allowed an understanding of the complex genetic makeup and gene functions in the major bioprocesses of the Brassicales, especially Brassica oil crops. Here, we provide an overview on the contributions of these technologies in improving the major traits of B. napus and discuss their potential use to accomplish new improvement targets.

Pollen-Specific Protein PSP231 Activates Callose Synthesis to Govern Male Gametogenesis and Pollen Germination.

Spatiotemporally regulated callose deposition is an essential, genetically programmed phenomenon that promotes pollen development and functionality. Severe male infertility is associated with deficient callose biosynthesis, highlighting the significance of intact callose deposition in male gametogenesis. The molecular mechanism that regulates the crucial role of callose in production of functional male gametophytes remains completely unexplored. Here, we provide evidence that the gradual upregulation of a previously uncharacterized cotton (Gossypium hirsutum) pollen-specific SKS-like protein (PSP231), specifically at the post pollen-mitosis stage, activates callose biosynthesis to promote pollen maturation. Aberrant PSP231 expression levels caused by either silencing or overexpression resulted in late pollen developmental abnormalities and male infertility phenotypes in a dose-dependent manner, highlighting the importance of fine-tuned PSP231 expression. Mechanistic analyses revealed that PSP231 plays a central role in triggering and fine-tuning the callose synthesis and deposition required for pollen development. Specifically, PSP231 protein sequesters the cellular pool of RNA-binding protein GhRBPL1 to destabilize GhWRKY15 mRNAs, turning off GhWRKY15-mediated transcriptional repression of GhCalS4/GhCalS8 and thus activating callose biosynthesis in pollen. This study showed that PSP231 is a key molecular switch that activates the molecular circuit controlling callose deposition toward pollen maturation and functionality and thereby safeguards agricultural crops against male infertility.