Metabolic pathways engineering for drought or/and heat tolerance in cereals.

Drought (D) and heat (H) are the two major abiotic stresses hindering cereal crop growth and productivity, either singly or in combination (D/+H), by imposing various negative impacts on plant physiological and biochemical processes. Consequently, this decreases overall cereal crop production and impacts global food availability and human nutrition. To achieve global food and nutrition security vis-a-vis global climate change, deployment of new strategies for enhancing crop D/+H stress tolerance and higher nutritive value in cereals is imperative. This depends on first gaining a mechanistic understanding of the mechanisms underlying D/+H stress response. Meanwhile, functional genomics has revealed several stress-related genes that have been successfully used in target-gene approach to generate stress-tolerant cultivars and sustain crop productivity over the past decades. However, the fast-changing climate, coupled with the complexity and multigenic nature of D/+H tolerance suggest that single-gene/trait targeting may not suffice in improving such traits. Hence, in this review-cum-perspective, we advance that targeted multiple-gene or metabolic pathway manipulation could represent the most effective approach for improving D/+H stress tolerance. First, we highlight the impact of D/+H stress on cereal crops, and the elaborate plant physiological and molecular responses. We then discuss how key primary metabolism- and secondary metabolism-related metabolic pathways, including carbon metabolism, starch metabolism, phenylpropanoid biosynthesis, γ-aminobutyric acid (GABA) biosynthesis, and phytohormone biosynthesis and signaling can be modified using modern molecular biotechnology approaches such as CRISPR-Cas9 system and synthetic biology (Synbio) to enhance D/+H tolerance in cereal crops. Understandably, several bottlenecks hinder metabolic pathway modification, including those related to feedback regulation, gene functional annotation, complex crosstalk between pathways, and metabolomics data and spatiotemporal gene expressions analyses. Nonetheless, recent advances in molecular biotechnology, genome-editing, single-cell metabolomics, and data annotation and analysis approaches, when integrated, offer unprecedented opportunities for pathway engineering for enhancing crop D/+H stress tolerance and improved yield. Especially, Synbio-based strategies will accelerate the development of climate resilient and nutrient-dense cereals, critical for achieving global food security and combating malnutrition.

Integration of transcriptome and metabolome analyses reveals key lodging-resistance-related genes and metabolic pathways in maize.

Stalk lodging, or breakage of the stalk at or below the ear, is one of the vital factors causing substantial yield losses in maize (Zea mays. L). Lodging affects maize plants' physiological and molecular processes, eventually impacting plant growth and productivity. Despite this known fact, few researchers have investigated the genetic architecture underlying lodging in maize. Herein, through integrated transcriptome, metabolome, and phenotypic analyses of stalks of three diverse hybrid cultivars (highly resistant JNK738, mildly resistant JNK728, and lowly resistant XY335) at the tasseling (10 days to silking, 10 DTS) stage, we identified key genes and metabolic pathways modulating lodging resistance in maize. Based on the RNA-Seq analysis, a total of 10093 differentially expressed genes (DEGs) were identified from the comparison of the three varieties in pairs. Additionally, key lodging resistance-related metabolic pathways were obtained by KEGG enrichment analysis, and the DEGs were found predominantly enriched in phenylpropanoid and secondary metabolites biosynthesis pathways in the L_vs._H and M_vs._H comparison groups. Moreover, K-means analysis clustered the DEGs into clear and distinct expression profiles for each cultivar, with several functional and regulatory genes involved in the cell wall assembly, lignin biosynthetic process and hormone metabolic process being identified in the special clusters related to lodging resistance. Subsequently, integrating metabolome and transcriptome analyses revealed nine key lignin-associated metabolites that showed different expression trends in the three hybrid cultivars, among which L-phenylalanine and p-coumaric acid were regarded as differentially changed metabolites (DCMs). These two DCMs belonged to phenylalanine metabolism and biosynthesis pathways and were also supported by the RNA-Seq data. Furthermore, plant hormone signal transduction pathway-related genes encoding auxin, abscisic acid, jasmonates, and salicylic acid were differentially expressed in the three comparisons of lodging resistance, indicating these DEGs were valuable potential targets for improving maize lodging resistance. Finally, comparative physiological and qRT-PCR analyses results supported our transcriptome-based findings. Our research not only provides a preliminary theoretical basis and experimental ideas for an in-depth study of the regulatory networks involved in maize lodging resistance regulation but also opens up new avenues for molecular maize stalk lodging resistance breeding.

Small G Protein StRab5b positively regulates potato resistance to Phytophthora infestans.

Rabproteins are the largest members of the small G protein family and are widely distributed in eukaryotes. It comprises eight subfamilies and is responsible for regulating vesicle transport, plant growth and development, and biotic and abiotic stress responses. In this study, the small G protein gene StRab5b was cloned from potato, and its biological information, expression profile and induced expression level, overexpression and gene silencing were examined on regulating potato resistance to Phytophthora infestans using PCR, qPCR and Virus-induced gene silencing (VIGS). Our results indicate that the amino acid of StRab5b shows the highest and lowest homology with NbRab5b in N. benthamiana and StRab in potato respectively. StRab5b expression varied among different potato tissues and varieties, and was induced by P. infestans infection. Transiently ectopic expression of StRab5b in N. benthamiana enhanced its resistance to P. infestans, whereas, silencing of StRab5b and its homologous gene facilitated pathogen infection in potato and N. benthamiana respectively. Furthermore, stable expression of the StRab5b gene in potatoes enhanced its redox-stress response capacity, as manifested by the accumulation of H2O2 in infected leaves and subsequent increase in the activity and expression of ROS scavenging enzymes, thereby attenuating the development of P. infestans and ultimately reducing the lesions on infected potato leaves. In addition, the LOX gene transcripts and JA level were upregulated rapidly after inoculation with P. infestans. Collectively, our results suggest that StRab5b positively regulates the resistance against potato late blight (PLB) via JA-mediated defense signaling pathway.

StRac1 plays an important role in potato resistance against Phytophthora infestans via regulating H2O2 production.

ROP GTPases (Rho-related GTPases from plant), a unique subgroup of the Rho family in plants, is a group of key regulators of different signaling pathways controlling plant growth and development, cell polarity and differentiation, and plant response against biotic and abiotic stresses. The present study determined the potential regulatory mechanism of potato ROP GTPase (StRac1) against Phytophthora infestans (P. infestans) infection. Protein secondary structure analysis indicated that StRAC1 is a Rho GTPase. The expression level of StRac1 was variable in different tissues of potato, with the highest expression in young leaves of both Shepody and Hutou potato varieties. After challenging with P. infestans, the expression level of StRac1was higher in resistance varieties Zihuabai and Longshu 7 than in susceptible varieties Shepody and Desiree. StRAC1 fusion with GFP subcellularly localized at the plasma membrane (PM) in tobacco epidermal cells. The potato with transient or stable over-expression of CA-StRac1 (constitutively active form of StRac1)exhibited a dramatic enhancement of its resistance against P. infestans infections. The increased resistance level in transgenic potato was accompanied with elevated H2O2 levels. Importantly, silencing StRac1 via virus-induced gene silencing (VIGS) in potato resulted in higher susceptibility to P. infestans infection than in control plants. In summary, our data reveal that StRac1 regulates potato resistance against P. infestans via positively modulating the accumulation of H2O2.

[Construction, Expression of hERα-LBD Prokaryotic Vector and the Activity of Expressed Protein].

OBJECTIVES: To construct the prokaryotic expression system of estrogen receptor α ligand bingding domain (hERα-LBD) and to evaluate the estrogen receptor ligand binding activity of the expressed protein. METHODS: hERα -LBD was amplicated from the plasmid of hERα -LBD by PCR, the identified PCR product was ligated with pGEM-T-easy vector to generate pGM-T-hERα -LBD. After the confirmation, the hERα -LBD fragments were obtained by enzyme digestion and inserted into pET-28a. The expression vectors were expressed in E.Coli to produce hERα-LBD protein. We mixed the hERα-LBD protein and estradiol and bovine serum albumin conjugated antigens (E2-BSA), then evaluated the binding activity of hERα-LBD by electrophoresis. RESULTS: The amplified fragment was about 1.9 kb, which was in agreement with the expected target fragment. Recombinant plasmid of pGM-T-hERα -LBD was confirmed by enzyme digestion and sequencing, then pET-28a(+)-hERα -LBD was constructed successfully. The expressed hERα-LBD protein in E.Coli was observed and the expression amount was 250 mg/L after affinity chromatography purification. hERα-LBD was confirmed to had estrogen binding activity by electrophoresis. CONCLUSIONS: The prokaryotic expression system of pET-28a(+)-hERα -LBD was successfully constructed, and hERα-LBD had the activity of binding.

Synthesis, Antifungal Activity and Structure-Activity Relationships of Novel 3-(Difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic Acid Amides.

A series of novel 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxylic acid amides were synthesized and their activities were tested against seven phytopathogenic fungi by an in vitro mycelia growth inhibition assay. Most of them displayed moderate to excellent activities. Among them N-(2-(5-bromo-1H-indazol-1-yl)phenyl)-3-(difluoro-methyl)-1-methyl-1H-pyrazole-4-carboxamide (9m) exhibited higher antifungal activity against the seven phytopathogenic fungi than boscalid. Topomer CoMFA was employed to develop a three-dimensional quantitative structure-activity relationship model for the compounds. In molecular docking, the carbonyl oxygen atom of 9m could form hydrogen bonds towards the hydroxyl of TYR58 and TRP173 on SDH.