Wild Lines Research Articles

Sodium Chloride Tolerance during Germination and Seedling Stages of Tomato (Solanum lycopersicum L.) Lines Native to Mexico

Tomato is considered moderately sensitive to salinity, which detracts from the quality and yield of its fruit; therefore, wild populations have been used as a genetic resource. The aim of this research was to identify lines derived from wild tomato populations with tolerance to salinity during the germination and seedling stages. During germination, 52 wild lines and 2 commercial hybrids (Imperial®, Reserva®) were subjected to treatment with 150 mM and 0 mM NaCl and evaluated. The test was carried out for 20 days in a germination chamber with constant darkness, a temperature of 25 ± 2 °C and relative humidity conditions of 80 ± 4%. At the seedling stage, 22 wild tomato lines with the best performance in the germination test and 2 commercial hybrids (Imperial®, Topanga®) were evaluated for 12 days in a floating raft system. Concentrations of 175 mM and 0 mM of NaCl were used. During germination, the saline condition decreased the germination percentage (65.2%), speed of germination (88.2%), steam length (72.5%), root length (46.56%), number of normal plants (59.5%), stem dry matter (68.78%), root dry matter (61.99%), and total dry matter (67.1%). At the seedling stage, this condition decreased (p < 0.05) the aerial part dry matter (46.37%), leaf area (59.35%), root length (42.43%), final plant height (40.24%), and growth rate (71.42%). Seventeen tolerant genotypes were identified in one of the two developmental stages, while one genotype showed tolerance in both stages. These results indicate that there are different response mechanisms in each developmental stage. Native tomatoes play an important role in the identification of tolerant genotypes since they can be used as genetic resources for obtaining commercial genotypes with salt tolerance.

Identification of allelic relationship and translocation region among chromosomal translocation lines that leads to less-seed watermelon.

Less-seed and seedless traits are desirable characteristics in watermelon (Citrullus lanatus). Hybridization between watermelon chromosomal translocated lines and wild lines significantly reduced seed counts in the hybrid fruits, approaching even seedless. However, the allelic relationships and the chromosomal translocation breakpoints from different sources are unclear, which limits their utility in breeding practices. This study focused on three groups of chromosomal translocation materials from different sources and conducted inheritance and allelic relationship analysis of translocation points. The results from third-generation genome sequencing and fluorescence in situ hybridization (FISH) revealed that the specific translocations in the naturally mutated material MT-a involved reciprocal translocations between Chr6 and Chr10. The Co60γ radiation-induced mutant material MT-b involved reciprocal translocations between Chr1 and Chr5, Chr4 and Chr8. The Co60γ radiation-induced mutant material MT-c involved complex translocations among Chr1, Chr5, and Chr11. Cytological observation showed that heterozygous translocation hybrids showed chromosomal synapsis abnormalities during meiotic diakinesis. Further, dominant and codominant molecular markers were developed on both sides of the translocation breakpoints, which could facilitate rapid and efficient identification of chromosome translocation lines. This study provides technical guidance for utilizing chromosomal translocation materials in the development of less-seed watermelon varieties.

Exploration of unique starch physicochemical properties of novel buckwheat lines created by crossing Golden buckwheat and Tatary buckwheat

Buckwheat is considered as a healthy cereal food, and it is essential to cultivate new buckwheat lines with good starch physicochemical properties for both consumers and food producers. Six novel buckwheat (Duoku, Dk) were generated by crossing of Golden buckwheat and Tatary buckwheat, and their kernel appearance properties and starch physicochemical properties were analyzed together with one domestic line (Cimiqiao) and one wild line (Yeku). The results showed that Dk samples had better appearance properties than two control samples. The Dk samples showed lower amylose content, similar amylopectin molecular structure and chain length distributions, and larger starch granules compared with Cimiqiao. The digestion results showed that two Dk samples: Dk6 & Dk9 had high resistant starch content; while the other two Dk samples: Dk37 & Dk38 had a steady glucose releasing rate. The Dk samples also showed high gelatinization temperature, indicating they were good raw materials for producing glass noodle. This study proved that Dk buckwheat had unique starch physicochemical properties, and could be used as new food materials in the future.

Features of the development of a unique legume-rhizobium symbiosystem under conditions of metal-induced stress

The development of legume-rhizobium symbiosystems based on the mutant pea genotype SGECdt (Pisum sativum L.) and its wild line SGE grown on a medium supplemented with toxic concentrations of Cd and Co was evaluated under the conditions of a summer greenhouse experiment. Plants were inoculated with a consortium of endomycorrhizal fungus Glomus sp.1Fo, nodule bacteria Rhizobiom leguminosarum bv. viciae and associative ACC-utilizing rhizobacteria Variovorax paradoxus 5C-2. Heavy metals significantly inhibited the growth of uninoculated and inoculated wild line SGE plants compared to SGECdt plants. The content of Cd in the shoots of plants of both genotypes increased in the presence of a toxic concentration of Co, while the latter, on the contrary, decreased. The same effect persisted after inoculation with microorganisms. The introduction of microbes leveled the toxic effect of heavy metals and increased the biomass in both pea genotypes in the absence of the introduction of heavy metals. The microbial consortium also contributed to an increase in the transport of biophilic microelement antagonists to the aerial organs of the pea. In general, based on the results of fractal calculation, despite growth inhibition, the wild line showed higher values of the degree of organization of microelements inside shoots and seeds than the mutant genotype. The stagnation or decrease in the microelement bioconsolidation indices in the homeostasis of shoots of the mutant line can probably be interpreted by the redistribution of the supply of food sources between the partners of the symbiostem, in favor of microsymbionts. An increase in the indicators of the bioconsolidation index in seeds can be considered a positive effect, since the best mobilization of trace elements in their cotyledons, in addition to increasing the germination energy of the offspring, will favorably affect the increase in the adaptive potential of plants.

A 43 Bp-Deletion in the F3'H Gene Reducing Anthocyanins Is Responsible for Keeping Buds Green at Low Temperatures in Broccoli.

Most broccoli cultivars or accessions exhibit green buds under appropriate growth conditions, which turn purple at cold temperatures. However, certain cultivars consistently maintain green buds both during normal growth and at cold temperatures. In this study, we used BSA-seq (bulked segregation analysis-sequencing), along with fine mapping and transcriptome analysis to identify a candidate gene (flavonoid 3'-hydroxylase, F3'H) responsible for reducing anthocyanin accumulation in the mutant GS and HX-16 broccoli (Brassica oleracea L. var. italica), which could retain green buds even at low temperatures. A 43-bp deletion was detected in the coding sequence (CDS) of the F3'H gene in HX-16 and the mutant GS, which significantly decreased F3'H expression and the accumulation of cyanidin and delphinidin in the mutant GS. Furthermore, the expression of F3'H was upregulated at low temperatures in the wild line PS. Our results demonstrated the efficacy of utilizing the 43-bp InDel (Insertion-Deletion) in predicting whether buds in B. oleracea L. will turn purple or remain green at cold temperatures across forty-two germplasm materials. This study provides critical genetic and molecular insights for the molecular breeding of B. oleracea and sheds light on the molecular mechanisms underlying the effect of low temperatures on bud color in broccoli.

Генотипический скрининг устойчивости зернобобовых культур к воздействию тяжелых металлов на основании нейронного профилирования экссудации их аминокислот

Abstract. The purpose of the article was to assess the change in the level of structural organization of the amino acid profile of root exometabolites of various resistance genotypes of peas under the influence of metal-induced stress. Research method. Plants were grown under sterile controlled conditions (climatic chamber) with the addition of toxic concentrations, cadmium, cobalt, zinc or mercury to the nutrient medium. The objects for the study were the Cd-sensitive genotype of the pea variety SGE, as well as the unique Cd-resistant mutant SGECDt created on its basis. Results. It has been established that all salt solutions of metals have an inhibitory effect on plant growth rates. At the same time, as expected, the addition of cadmium and cobalt to the medium had a greater effect on the decrease in the biomass of the wild line SGE than in the mutant genotype SGECDt. The latter reacted more to the introduction of toxic mercury into the environment. As for zinc, here, the inhibition of the biomass of both organs in the two genotypes was equivalent. The addition of all toxicants to the medium led to an increase in the total yield of all amino acids. In the SGECDt mutant, this manifested itself to a greater extent, which is associated with certain aspects of metal detoxification in this genotype in plant tissues. Cluster analysis made it possible to separate the ratios of the amino acid profile obtained on zinc and mercury in both genotypes into a separate independent group. The results of calculations using a neural network confirmed the resistance of the mutant to Cd and Co ions, and sensitivity to Zn and Hg. The wild line was found to be resistant to the selected concentration of Co. Scientific innovation. The mathematical model, designed on the basis of the collected amino acid exudation data array, makes it possible, on the basis of a matrix of correlation ratios, to predict the yield of absolutely dry plant protein biomass and to primary screening the adaptive lability of various varieties under metal-induced stress.

Dynamics of growth and accumulation of heavy metals of Pisum Sativum L. grown on polluted soil (vegetative experience)

The paper presents the results of studies of the effect of heavy metal salts (15 mg/kg CdCl2 and/or 50 mg/kg CoCl2) contamination of soddy-podzolic soil on the adaptive potential and phytoremediation activity of two pea genotypes contrasting in resistance: the cadmium-resistant mutant SGECDt and its wild line SGE. The experiment was carried out under laboratory conditions while maintaining a stable internal microclimate and an optimal degree of insolation in the room. The duration of the experiment was 85 days. A study of the dynamics of morphometric parameters, using the example of shoot and root biomass, showed that the SGECDt mutant demonstrated better resistance to toxicants: on average, the decrease in shoot biomass compared to control was 28.2%, and 5.5% for roots. Whereas on wild lines, the rates were 31.8% on shoots and 21.8% on roots, respectively. As expected, the tolerance of the mutant was best manifested in the variant with the introduction of cadmium ions. Thanks to mass spectrometric analysis, it was found that the content of cadmium in dry biomass of shoots increased with an increase in biomass in both genotypes. Moreover, in the mutant, it significantly exceeded the concentration of pollutant accumulation in the wild type, and practically did not change, starting from 30 days with separate or combined introduction of metals, and until the end of the growing season - 20-25 µg of dry biomass per gram of shoot. The content of cobalt, on the contrary, decreased in ontogenesis phases, with a slight superiority in metal fixation by the mutant, starting from 45 days after inoculation. With the simultaneous entry of both metals into the soil solution, synergism of action was not observed. The content of cobalt decreased even more in plants, while cadmium, on the contrary, increased. It can be concluded that the ion-exchange reactions between these pollutants differed in interactions in the "soil-plant" system by the type of antagonism. Among the optimal period for harvesting contaminated products for its further processing (phytomining), one can single out the budding phase - the beginning of flowering (45 days). During this period, the mutant plants simultaneously perform well detoxification (phytosanation) and active accumulation of atmospheric nitrogen in the soil.

Quantitative genetic analysis of attractiveness of yeast products to Drosophila

The interaction between two model organisms, the fruit fly Drosophila melanogaster, and the budding yeast Saccharomyces cerevisiae, is widely perceived as mutually beneficial. The bridge of this biological connection is the volatile compounds from yeast fermentation, which can be attractive to the flies. The generation of volatile molecules derives from complex metabolism and different yeast will generate different combinations of volatiles. The attractiveness to fruit flies can therefore be considered a complex trait. In this study, six strains from SGRP were selected for fermenting commercial grape juice. The attractiveness of these ferments to a wild fruit fly line was then tested. Flies prefer the ferments of the Wine European (WE) strain over the North America (NA) strain. The preference assay was then applied to ferments of genotyped F1 progeny of WE crossed to NA for QTL mapping of genetic variation in attractiveness. GC-MS profiling of all ferments demonstrated that NA could be easily distinguished from other strains. 15 compounds were further quantified and the concentrations in F1 progeny ferments were correlated with genotypes using our r/QTL pipeline. Candidate genes from QTL mapping were tested by reciprocal hemizygosity analysis, validating the impact of two genes, PTC6 and YFL040W. The alleles of these validated genes from NA has better effect on the preference assay than the allele from WE, which is can be considered as antagonistic QTLs. The identification variation in these two genes underpinning metabolic differences, demonstrates how WE and NA have different attraction to one wild fruit fly line.

Abstract 6086: Clonal biallelic inactivation of NF2 is an evolutionary bottleneck that exposes a vulnerability to CDK7 inhibition in mesothelioma

Abstract Background: Mesothelioma, a cancer caused by asbestos is lethal and lacks molecularly targeted therapy. The tumor suppressor NF2 constitutes a frequent, positively selected somatic alteration warranting synthetic lethal approaches to target this driver. NF2 inactivation drives nuclearization of YAP to mediate TEAD dependent oncogenic transcriptional program. YAP is stabilized by CDK7 through phosphorylation of S169/S128/S90, inhibiting the E3 ligase complex CDL4DCAF12. We hypothesized that biallelic NF2 inactivation would be vulnerable to augmentation of CRL4DCAF12 through inhibition of CDK7. Methods: 50 Patients (the MEDUSA cohort) undergoing routine extended pleurectomy decortications consented to have multiregional sampling of their mesotheliomas at resection at 4-5 stereotyped locations, followed by multiregional whole exome sequencing (mWES 200x), phylogenetic deconvolution and transfer learning to examine evolutionary trajectories. Bulk RNA sequencing enabled gene set enrichment analysis, and spatial YAP phenotyping using tissue microarrays were conducted on the mWES profiled cohort. Primary cell lines were generated from a subset of mesotheliomas and whole exome sequenced. Results: NF2 exhibited biallelic inactivation involving copy number/mutation allelic heterogeneity in 42% of mesotheliomas. Double hits were predominantly clonal. Hippo signalosome exhibited additional clonal somatic alterations in 26% of mesotheliomas, with biallelic inactivation being observed in LATS2 (8%) and LATS1 (4%). Transfer learning identified NF2 inactivation as a predominantly secondary clonal event, constituting an evolutionary bottleneck. Random forest analysis revealed YAP dependent transcription, epithelial mesenchymal transition and spindle checkpoint transcriptional signature enrichments in NF2 double hit mesotheliomas. We conducted drug screening of NF2 wild type versus inactivated primary mesothelioma cell lines, which revealed selective sensitivity to CDK7 inhibitors THZ1 and YKL-5-124 in NF2 inactivated but not wild type MEDUSA cell lines. Conclusions: NF2 biallelic inactivation constitutes an evolutionary bottleneck during early mesothelioma evolution, associated with a specific vulnerability to CDK7 highlighting a potential path to clinical stratified therapy. Citation Format: Essa Y. Baitei, Min Zhang, Qianqian Sun, Jin-Li Luo, Philip Zhang, Tamihiro Kamata, James Harber, Aleksandra Bzura, Peter W. Jordan, Charlotte Poile, Alan Dawson, Apostolos Nakas, Cathy Richards, Hongji Yang, Ed Hollox, Dean A. Fennell. Clonal biallelic inactivation of NF2 is an evolutionary bottleneck that exposes a vulnerability to CDK7 inhibition in mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6086.

Abstract 178: Multidimensional hydrogel model to benchmark progression of temozolomide-resistant glioblastoma

Abstract Introduction: Glioblastoma (GBM) is the most common and lethal form of brain cancer with a median survival of only 12 months. Due to challenges associated with surgical removal, resistance to chemo- and radiotherapy, and highly invasive nature of the tumor, GBM typically recurs rapidly near the primary tumor site, and the only widely-used therapeutic agent against GBM, temozolomide (TMZ), is merely effective. Previously, we have shown that a methacrylamide-functionalized gelatin (GelMA) hydrogel platform can support addition of brain-mimetic molecules and formation of vascular networks to study GBM behavior. In this study, we assess GBM response to TMZ using a unique set of isogenically matched GBM cell lines (8MGBA-WT vs. 8MGBA-TMZres; 42MGBA-WT vs. 42MBGA-TMZres) that exhibit TMZ responsiveness (WT) or resistance (TMZres) along with disparate proliferative and invasive patterns in two-dimensional culture. This study seeks to define changes in invasion and proliferation in response to TMZ within three-dimensional gelatin hydrogels. Methods: 8MGBA-WT, 8MGBA-TMZres, 42MGBA-WT, and 42MBGA-TMZres cell lines were cultured in DMEM with 10% FBS (100 μM TMZ continuously applied to 8MGBA-TMZres). Cells were encapsulated in GelMA hydrogels and cultured for 8 days with daily media change. After 24 hours, TMZ doses between 0 - 500 μM were applied to the gels and maintained for 7 days with daily media change. Growth rate inhibition (GR) statistics were determined after 7 days of culture. Relative cell count was assessed with the alamarBlue assay. GBM cell spheroids incapsulated in GelMA hydrogels were generated to measure relative invasion of wild type (WT) vs. TMZres lines. Spheroid outgrowth area was quantified with image analysis. Results: We observed changes in proliferative and invasive phenotypes between the cell lines consistent with 2D cell culture. Notably, 42MGBA-TMZres cells exhibited proliferative advantage, while 8MGBA-TMZres exhibited invasive advantage. GR analysis revealed apparent differences between WT and TMZres lines with the following GR50 values: 41 μM, 8MGBA-WT; 150 μM, 8MGBA-TMZres; 77 μM, 42MGBA-WT; 339 μM, 42MGBA-TMZres. These results provide insight into potential common pathways between the “go” or “grow” phenotypes that can be used as therapeutic targets for GBM. Conclusions: We have shown that a GelMA hydrogel platform can be used to assess GBM response to TMZ in 3D culture. This model provides insight into rapid tumor cell proliferation and diffusive local invasion. Further, this platform will be advanced to include a previously-developed model of GBM perivascular niche; this next-generation model will be used to benchmark the effect of perivascular niche on GBM therapeutic resistance and invasion. Hence, ongoing experiments aim to develop a vascularized hydrogel model of GBM to benchmark new TMZ variants hypothesized to overcome perivascular-induced resistance. Citation Format: Viktoriia Kriuchkovskaia, Brendan A. Harley, Rebecca B. Riggins. Multidimensional hydrogel model to benchmark progression of temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 178.

Abstract 5317: A novel approach to the treatment of ATRA/ATO resistant acute promyelocytic leukemia

Abstract Acute Promyelocytic Leukemia (APL) is characterized by proliferation of immature promyelocytes. Combination therapy using retinoic acid (RA) and arsenic trioxide (ATO) is considered standard treatment in the clinical environment. Resistance to both RA and ATO is associated with an increased risk of mortality. In addition, relapse occurs in approximately 10-20% of patients undergoing treatment for APL. Growth Hormone Releasing Hormone (GHRH) is a hypothalamic peptide that stimulates the release of Growth Hormone from the anterior pituitary. Our previous studies have demonstrated that human AML cell lines (K-562, THP-1, and KG-1a) display the GHRH receptor (GHRH-R). Incubation with GHRH-R antagonist MIA-602, inhibited proliferation and induced apoptosis. The purpose of this study is to examine the effects of GHRH-R antagonist MIA-602, on the NB4 Human Promyelocytic Leukemia cell line and its ATRA and ATO resistant sub-clone. NB4 cells expressed both GHRH-R and SV1 variants via western blot analysis. Wild type NB4 cells, as well as NB4 cells resistant to both ATRA and ATO (NB4-RAA), were cultured in suspension in RPMI and 10% fetal calf serum. NB4-RAA were established in Dr. Jimenez's lab from the wild type NB4 parent cell line. NB4 and NB4-RAA were plated in triplicate on multi-wells at 50,000 cells/mL. Cell lysates were prepared after incubation with 0.05-5 µmol/L of MIA-602. Cell viability was measured at 24 and 48h. Flow cytometry was also performed on the naïve NB4 parent cell line in addition to NB4-RAA to assess expression of CD-56 the cell surface marker. Upregulation of CD-56, also known as Neural cell adhesion molecule 1 (NCAM-1), has been shown to confer drug resistance in AML and is an important prognostic marker in APL. The viability of both cell lines decreased to similar levels at 24 h and 48 h when exposed to concentrations of MIA-602 higher than 0.05 μmol/L (p < 0.05). A significant decrease in cell viability was seen above 0.5 μmol/L after 48h. No viable cells were found in either cell line after 48h when exposed to 5 μmol/L of MIA-602. No difference in cell viability was found between naïve NB4 and resistant NB4-RAA when exposed to the same concentrations of MIA-602. MIA-602 induced significant changes in the expression level of many death-related genes including CASP9 (>1.5-fold; P<0.05). Results of flow cytometry revealed significantly increased expression of CD-56 (>5.8-fold; p,0.05) in NB4-RAA cells as compared to the parent cell line. These results indicate that resistance to ATRA and ATO in APL cells does not confer subsequent resistance to MIA-602. As a result, MIA-602 targets a pathway that is distinct from that of ATRA & ATO. More importantly, upregulation of pro-apoptotic genes by MIA-602 indicates an alternative pathway elicited by the GHRH-R antagonist in the setting of resistant APL cells. Our results indicate that the resistance to frontline APL therapy can be effectively overcome by using MIA-602. Citation Format: Ravinder S. Chale, Stephanie M. Almeida, Ivan Jozic, Andrew V. Schally, Joaquin J. Jimenez. A novel approach to the treatment of ATRA/ATO resistant acute promyelocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5317.

Abstract 2670: ERAS-601, a potent allosteric inhibitor of SHP2, synergistically enhances the efficacy of sotorasib/adagrasib and cetuximab in NSCLC, CRC, and HNSCC tumor models

Abstract ERAS-601 is a potent, small molecule allosteric inhibitor of wildtype SHP2, a non-receptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene. ERAS-601 inhibits wild type SHP2 biochemically with an IC50 of 4.6 nM and demonstrates selectivity across panels of 300 kinases and 12 phosphatases. SHP2 mediates upstream receptor tyrosine kinase (RTK) signaling via its phosphatase-mediated regulation of guanine nucleotide exchange factors (GEFs). ERAS-601 inhibits the SHP2 dependent cycling of KRAS from the inactive GDP-bound state to the active GTP-bound state and demonstrates anti-proliferative activity in KRASG12C and EGFR amplified cell lines. The combination of upstream blockade of RAS-GTP cycling by ERAS-601 with inhibition of KRASG12C by a selective KRASG12C inhibitor synergistically inhibits cellular proliferation in multiple KRASG12C mutated human cancer cell lines. The combinations of ERAS-601 with KRASG12C inhibitors achieve tumor growth inhibition that is superior to the respective ERAS-601 and KRASG12C monotherapies in NSCLC and CRC CDX and PDX tumor models. Similarly, the combination of ERAS-601 with an EGFR antibody, cetuximab, inhibits oncogenic RAS/MAPK signaling as measured by pERK1/2 and enhances the anti-proliferative activity of cetuximab in RAS/RAF wild type CRC and HPV-negative HNSCC cell lines. The combination of ERAS-601 with cetuximab achieves tumor growth inhibition that is superior to respective ERAS-601 and cetuximab monotherapies in RAS/RAF wild type CRC and HPV-negative HNSCC CDX and PDX tumor models. These nonclinical data support the clinical development of ERAS-601 in combination with a KRASG12C inhibitor in NSCLC and CRC tumors, and ERAS-601 in combination with cetuximab in RAS/RAF wild type CRC as well as HPV-negative HNSCC tumors. Both combinations are being studied in ongoing clinical studies (HERKULES-2, NCT04959981; FLAGSHP-1, NCT04670679). Citation Format: Leenus Martin, Roopal Patel, Jingchuan Zhang, Robin Nevarez, Taylor Congdon, Les Brail, Robert Shoemaker. ERAS-601, a potent allosteric inhibitor of SHP2, synergistically enhances the efficacy of sotorasib/adagrasib and cetuximab in NSCLC, CRC, and HNSCC tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2670.

Role of LOC_Os01g68450, Containing DUF2358, in Salt Tolerance Is Mediated via Adaptation of Absorbed Light Energy Dissipation.

Salt stress affects plant growth and productivity. In this study we determined the roles of eight genes involved in photosynthesis, using gene co-expression network analysis, under salt-stress conditions using Arabidopsis knockout mutants. The green area of the leaves was minimum in the at1g65230 mutant line. Rice LOC_Os01g68450, a homolog of at1g65230, was ectopically expressed in the at1g65230 mutant line to generate revertant lines. Under salt stress, the revertant lines exhibited significantly higher net photosynthesis rates than the at1g65230 mutant line. Moreover, the operating efficiency of photosystem II (PSII) and electron transport rate of the revertant lines were higher than those of the wild type and at1g65230 mutant line after 10 days of exposure to salt stress. After this period, the protein PsbD–the component of PSII–decreased in all lines tested without significant difference among them. However, the chlorophyll a and b, carotenoid, and anthocyanin contents of revertant lines were higher than those of the mutant line. Furthermore, lower maximum chlorophyll fluorescence was detected in the revertant lines. This suggests that LOC_Os01g68450 expression contributed to the salt tolerance phenotype by modifying the energy dissipation process and led to the ability to maintain photosynthesis under salt stress conditions.

Minerals, vitamins, protein and amino acids in wild Cicer species and pure line chickpea genotypes selected from a local population

Minerals, vitamins, protein and amino acids in wild Cicer species and pure line chickpea genotypes selected from a local population

Degradome comparison between wild and cultivated rice identifies differential targeting by miRNAs

BackgroundSmall non-coding (s)RNAs are involved in the negative regulation of gene expression, playing critical roles in genome integrity, development and metabolic pathways. Targeting of RNAs by ribonucleoprotein complexes of sRNAs bound to Argonaute (AGO) proteins results in cleaved RNAs having precise and predictable 5` ends. While tools to study sliced bits of RNAs to confirm the efficiency of sRNA-mediated regulation are available, they are sub-optimal. In this study, we provide an improvised version of a tool with better efficiency to accurately validate sRNA targets.ResultsHere, we improvised the CleaveLand tool to identify additional micro (mi)RNA targets that belong to the same family and also other targets within a specified free energy cut-off. These additional targets were otherwise excluded during the default run. We employed these tools to understand the sRNA targeting efficiency in wild and cultivated rice, sequenced degradome from two rice lines, O. nivara and O. sativa indica Pusa Basmati-1 and analyzed variations in sRNA targeting. Our results indicate the existence of multiple miRNA-mediated targeting differences between domesticated and wild species. For example, Os5NG4 was targeted only in wild rice that might be responsible for the poor secondary wall formation when compared to cultivated rice. We also identified differential mRNA targets of secondary sRNAs that were generated after miRNA-mediated cleavage of primary targets.ConclusionsWe identified many differentially targeted mRNAs between wild and domesticated rice lines. In addition to providing a step-wise guide to generate and analyze degradome datasets, we showed how domestication altered sRNA-mediated cascade silencing during the evolution of indica rice.

MtNOOT gene enhanced high productivity and economical characteristics of tomato (Lycopersicon esculentum)

Tomato (Lycopersicon esculentum) is the main vegetal crop that has tremendous popularity around the world. Medicago truncatula NOOT gene (Mt-NOOT) encodes a BTB/POZ-ankyrin repeat protein of the NONEXPRESSOR OF PR GENES1 (NPR1 family). It was introduced into Lycopersicon esculentum (Tomato) genome. The tomato plants that ectopically expressed Mt-NOOT obtained several favorable traits and fruit quality. Heteroblasty between the transgenic and the non-transgenic tomato leaves and flower architecture were used to distinguish transgenic and wild lines. Transgenic tomato plants accumulated a significant amount of phenolic compounds and plant pigmentations compared to the wild type. On the other hand, transgenic plants acquired a considerable amount of antioxidant such as CuZnSO superoxide dismutase (SOD), tomato Catalase (CAT), and tomato Cell wall-associated peroxidase (TPX1) than the wild type. Antioxidant high content together with the high content of phenolic compounds enabled the transgenic tomato fruits to gain not only edible benefits, but also a significant higher shelf-time, extended to six months more than the wild type stored at 25°C in dark and dry condition. Surprisingly, transgenic tomato fruits did not show any rotten process during long time storage as they did not acquire any contagious microorganism. Total fruit productivity in transgenic tomato was greater than the control with an estimated ratio of 84%.

Colony fitness increases in the honey bee at queen mating frequencies higher than genetic diversity asymptote

Across the eusocial Hymenoptera, a queen’s mating frequency is positively associated with her workers’ genetic diversity and colony’s fitness. Over 90% of a colony’s diversity potential is achieved by its mother’s tenth effective mating (me); however, many females mate at levels of me > 10, a zone we here call hyperpolyandry. We compared honey bee colony fitness at mating levels near and above this genetic diversity asymptote. We were interested in how hyperpolyandry affects colony phenotypes arising from both common tasks (brood care) and rare specialized tasks (parasite resistance). We used an unselected wild line of bees and a Varroa Sensitive Hygiene (VSH) line selected to resist the parasite Varroa destructor. Virgin queens were instrumentally inseminated to replicate the following queen/colony conditions: (1) VSH semen/low polyandry (observed mating number = mo = 9), (2) VSH semen/high polyandry (mo = 54), (3) wild type semen/low polyandry, or (4) wild semen/high polyandry. There was a positive effect of polyandry on brood survival, an outcome of common tasks, with highest values at mo = 54. There was an interaction between polyandry and genetics such that differences between genetic lines expressed only at mo = 54, with fewer mites in VSH colonies. These results are consistent with two hypotheses for the evolution of mating levels in excess of the genetic diversity asymptote: hyperpolyandry improves colony fitness by (1) optimizing genotype compositions for common tasks and (2) by capturing rare specialist allele combinations, resisting cliff-edge ecological catastrophes.Significance statementPolyandry is a female’s practice of mating with several males, storing their sperm, and using it to produce one or more clutches of genetically diverse offspring. In the social Hymenoptera, polyandry increases the genetic diversity and task efficiency of workers, leading to improved colony fitness. Over 90% of the increase in a colony’s diversity potential is achieved by its mother’s tenth mating; however, many females practice hyperpolyandry, a term we reserve here for mating levels above this genetic diversity asymptote. We show that a token of colony fitness arising from common tasks, brood survival, improves universally as one moves from sub- to hyperpolyandrous mating levels. However, a colony phenotype arising from a rare parasite resistance task is only expressed in the presence of the controlling alleles and under conditions of hyperpolyandry. These results suggest adaptive mechanisms by which hyperpolyandry could evolve.

Transcription Factor Pso9TF Assists Xinjiang Wild Myrobalan Plum (Prunus sogdiana) PsoRPM3 Disease Resistance Protein to Resist Meloidogyne incognita.

The root-knot nematode (Meloidogyne incognita) causes huge economic losses in the agricultural industry throughout the world. Control methods against these polyphagous plant endoparasites are sparse, the preferred one being the deployment of plant cultivars or rootstocks bearing resistance genes against Meloidogyne species. Our previous study has cloned one resistance gene, PsoRPM3, from Xinjiang wild myrobalan plum (Prunus sogdiana). However, the function of PsoRPM3 remains elusive. In the present study, we have investigated the regulatory mechanism of PsoRPM3 in plant defense responses to M. incognita. Our results indicate that fewer giant cells were detected in the roots of the PsoRPM3 transgenic tobacco than wild tobacco lines after incubation with M. incognita. Transient transformations of full-length and TN structural domains of PsoRPM3 have induced significant hypersensitive responses (HR), suggesting that TIR domain might be the one which caused HR. Further, yeast two-hybrid results revealed that the full-length and LRR domain of PsoRPM3 could interact with the transcription factor Pso9TF. The addition of Pso9TF increased the ROS levels and induced HR. Thus, our data revealed that the LRR structural domain of PsoRPM3 may be associated with signal transduction. Moreover, we did not find any relative inductions of defense-related genes PsoEDS1, PsoPAD4 and PsoSAG101 in P. sogdiana, which has been incubated with M. incognita. In summary, our work has shown the key functional domain of PsoRPM3 in the regulation of defense responses to M. incognita in P. sogdiana.

Abstract 2081: Transcriptional silencing of CDK18 in BRG1 mutant lung cancer

Abstract In ~10% of lung cancers, the BRG1 gene is mutated often causing a loss of function (LOF). BRG1 is a tumor suppressor that encodes a protein (SMARCA4), which is one of the mutually exclusive ATPase subunits of the Switch/Sucrose Non-fermentable (SWI/SNF) complex. The SWI/SNF complex functions to regulate transcription of many genes including tumor suppressor genes, through mediating chromatin remodeling and nucleosome repositioning by maintaining open chromatin and actively evicting polycomb repressive complexes (PRCs) from target genes and regions. Our previous research showed that BRG1 mutation occurs in approximately 9% of lung adenocarcinomas in smokers and 11% in smokers delineating BRG1 as a clinically relevant mutation. The goal of these studies is to elucidate the downstream effects of BRG1 LOF on gene expression. RNA sequencing of isogenic BRG1 wild type (WT) and knock out (KO) clones from malignant and transformed cell lines identified cell cycle control and chromosomal replication as the most significantly affected pathway. qRT-PCR was used to validate expression changes in 17 genes from this pathway in the WT and KO clones as well as wild type and mutant tumors lines. CDK18 was the most significantly affected target exhibiting a 78% reduction in expression (p<0.001) comparing wild type to mutant cell lines. Ongoing studies are investigating the epigenetic mechanisms leading to transcriptional repression of CDK18 that include cytosine DNA methylation and histone methylation of H3K27. In addition, functional assays (e.g., soft agar, cell migration) are evaluating the role of epigenetically repressed CDK18 in lung carcinogenesis and cell cycle control through siRNA knock down of CDK18 in wild type BRG1 cell lines and carcinogen transformed human bronchial epithelial cells. Together, these studies will provide new insight into the role of LOF of CDK18 in BRG1 mutant cancer. (Supported by P30 CA11800). Citation Format: Loryn Michelle Phillips, Christopher A. Johnston, Steven A. Belinsky, Mathewos Tessema. Transcriptional silencing of CDK18 in BRG1 mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2081.

Using some growth stimuli, a comparative study of salt tolerance in two tomatoes cultivars and a related wild line with special reference to superoxide dismutases and related micronutrients.

Salinity is a major global problem that threatens the agricultural sector, especially in areas that suffer from a shortage of water. It motivates ionic toxicity, osmotic and oxidative stresses, which greatly inhibits plant performances and crop productivites. However, micronutrients (MNs) or plant extracts, like germinated maize grain extract (gMGE), have been reported to minimize the effects of salt stress on plant growth and returns. Therefore, this study aimed at evaluating the influences of MNs or gMGE applied as foliar sprays on growth, physio-biochemical indices, and antioxidative system components in three genotypes of tomato plants stressed by 9 dS m−1 NaCl. This salinity level markedly increased Na+ content, lipid peroxidation, ion leakage, and markers related to oxidative stress (superoxide; O2•− and hydrogen peroxide; H2O2). Besides, marked increases in activities of enzymatic (especially different forms of superoxide dismutase; SODs) and non-enzymatic antioxidants and osmoprotectant compounds were also observed. In contrast, growth, photosynthetic capacity including hill reaction activity (HRA), K+/Na+ ratio, tissue cell integrity (e.g., cell water content and membrane stability), and K+ and MNs contents decreased significantly under stress. However, compared to MNs, gMGE significantly improved the activities of the antioxidative system components (particularly SODs) and osmoprotectants, which were reflected in reduced Na+ accumulation, lipid peroxidation, ion leakage, and oxidative stress. These results were coupled with remarkable elevations in photosynthetic capacity including HRA, K+/Na+ ratio, tissue cell integrity, K+ content, and MNs contents, all of which were reflected in the enhancement of plant growth. Compared to local tomato cultivars (e.g., Castle Rock and C10), the wild line “0043-1” had better results. The interaction of three factors; salt stress, promoters, and tomato genotypes was significant. The wild tomato line “0043-1” as the best salt-tolerant is a good candidate for implication in breeding programs for tolerance to salinity to produce salt-tolerant cultivars for use to maximize tomato growth and productivity in saline environments.