Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis).

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

Coffee Cell Suspensions as a Platform for Transient Gene Expression Analysis.

Coffea arabica L. is a crucial crop globally, but its genetic homogeneity leads to its susceptibility to diseases and pests like the coffee berry borer (CBB). Chemical and cultural control methods are difficult due to the majority of the CBB life cycle taking place inside coffee beans. One potential solution is the use of the gene cyt1Aa from Bacillus thuringiensis as a biological insecticide. To validate candidate genes against CBB, a simple, rapid, and efficient transient expression system is necessary. This study uses cell suspensions as a platform for expressing the cyt1Aa gene in the coffee genome (C. arabica L. var. Catuaí) to control CBB. The Agrobacterium tumefaciens strain GV3101::pMP90 containing the bar and cyt1Aa genes are used to genetically transform embryogenic cell suspensions. PCR amplification of the cyt1Aa gene is observed 2, 5, and 7 weeks after infection. This chapter describes a protocol that can be used for the development of resistant varieties against biotic and abiotic stresses and CRISPR/Cas9-mediated genome editing.

Screening and functional analysis of StMYB transcription factors in pigmented potato under low-temperature treatment.

MYB transcription factors play an extremely important regulatory role in plant responses to stress and anthocyanin synthesis. Cloning of potato StMYB-related genes can provide a theoretical basis for the genetic improvement of pigmented potatoes. In this study, two MYB transcription factors, StMYB113 and StMYB308, possibly related to anthocyanin synthesis, were screened under low-temperature conditions based on the low-temperature-responsive potato StMYB genes family analysis obtained by transcriptome sequencing. By analyzed the protein properties and promoters of StMYB113 and StMYB308 and their relative expression levels at different low-temperature treatment periods, it is speculated that StMYB113 and StMYB308 can be expressed in response to low temperature and can promote anthocyanin synthesis. The overexpression vectors of StMYB113 and StMYB308 were constructed for transient transformation tobacco. Color changes were observed, and the expression levels of the structural genes of tobacco anthocyanin synthesis were determined. The results showed that StMYB113 lacking the complete MYB domain could not promote the accumulation of tobacco anthocyanins, while StMYB308 could significantly promote the accumulation involved in tobacco anthocyanins. This study provides a theoretical reference for further study of the mechanism of StMYB113 and StMYB308 transcription factors in potato anthocyanin synthesis.

Unveiling the defensive role of Snakin-3, a member of the subfamily III of Snakin/GASA peptides in potatoes.

KEY MESSAGE: The first in-depth characterization of a subfamily III Snakin/GASA member was performed providing experimental evidence on promoter activity and subcellular localization and unveiling a role of potato Snakin-3 in defense Snakin/GASA proteins share 12 cysteines in conserved positions in the C-terminal region. Most of them were involved in different aspects of plant growth and development, while a small number of these peptides were reported to have antimicrobial activity or participate in abiotic stress tolerance. In potato, 18 Snakin/GASA genes were identified and classified into three groups based on phylogenetic analysis. Snakin-1 and Snakin-2 are members of subfamilies I and II, respectively, and were reported to be implicated not only in defense against pathogens but also in plant development. In this work, we present the first in-depth characterization of Snakin-3, a member of the subfamily III within the Snakin/GASA gene family of potato. Transient co-expression of Snakin-3 fused to the green fluorescent protein and organelle markers revealed that it is located in the endoplasmic reticulum. Furthermore, expression analyses via pSnakin-3::GUS transgenic plants showed GUS staining mainly in roots and vascular tissues of the stem. Moreover, GUS expression levels were increased after inoculation with Pseudomonas syringae pv. tabaci or Pectobacterium carotovorum subsp. carotovorum and also after auxin treatment mainly in roots and stems. To gain further insights into the function of Snakin-3 in planta, potato overexpressing lines were challenged against P. carotovorum subsp. carotovorum showing enhanced tolerance to this bacterial pathogen. In sum, here we report the first functional characterization of a Snakin/GASA gene from subfamily III in Solanaceae. Our findings provide experimental evidence on promoter activity and subcellular localization and reveal a role of potato Snakin-3 in plant defense.

Fate of transgenic soybean DNA and immune response of broilers fed genetically modified DP-3Ø5423-1 soybean.

Increased use of genetically modified (GM) plants in the food and feed industry has raised several concerns about the presence of unwanted genes in the food chain and potential associated health risks. In recent years, several studies have compared the nutrient contents of GM crops to conventional counterparts, and some have also tracked the fate of novel DNA fragments and proteins in the gastrointestinal (GIT) and their presence in several tissues. This study was conducted to investigate the fate of transgenic PHP19340A DNA fragment containing gm-fad2-1 (Soybean Event DP-3Ø5423-1) gene in digestive tract contents, blood, internal organs, and muscle tissues. The effects of feeding DP-3Ø5423-1 full-fat soybean meal (FFSBM) to broiler chickens on immune response and blood profiles were also evaluated on d 35. Day-old Ross 308 birds (n = 480) were randomly allocated to 24 floor pens in a 2 × 2 factorial arrangement with diet and gender as main factors. Birds were fed diets containing 20% of either DP-3Ø5423-1 or non-GM FFSBM for 35 d. Data were subjected to a 2-way ANOVA using the GLM procedure of JMP (Pro13). Based on PCR analysis, transgenic PHP19340A DNA fragment containing gm-fad2-1 gene was degraded throughout the digestive system to reach undetectable level in the cecal digesta. Moreover, there was no transgenic gene translocation to blood, organs, or muscle tissue. Feeding DP-3Ø5423-1 FFSBM to broilers had no effect on mRNA abundance of IL-1ß, IL-2, IL-6, IL-12B, IL-17A, IFNγ, TNFα, and NF-κB in the spleen or on blood profile. In conclusion, these findings indicate that the examined transgenic fragment in DP-3Ø5423-1 FFSBM progressively degraded in the GIT and did not translocate into blood or tissues. Along with the immune response and blood profile findings, it can be assumed that DP-3Ø5423-1 soybean is safe and unlikely to pose any health risks to broilers or consumers.

PgDDS Changes the Plant Growth of Transgenic Aralia elata and Improves the Production of Re and Rg3 in Its Leaves.

Aralia elata (Miq.) Seem is a medicinal plant that shares a common pathway for the biosynthesis of triterpenoid saponins with Panax ginseng. Here, we transferred the dammarenediol-II synthase gene from P. ginseng (PgDDS; GenBank: AB122080.1) to A. elata. The growth of 2-year-old transgenic plants (L27; 9.63 cm) was significantly decreased compared with wild-type plants (WT; 74.97 cm), and the leaflet shapes and sizes of the transgenic plants differed from those of the WT plants. Based on a terpene metabolome analysis of leaf extracts from WT, L13, and L27 plants, a new structural skeleton for ursane-type triterpenoid saponins was identified. Six upregulated differentially accumulated metabolites (DAMs) were detected, and the average levels of Rg3 and Re in the leaves of the L27 plants were 42.64 and 386.81 µg/g, respectively, increased significantly compared with the WT plants (15.48 and 316.96 µg/g, respectively). Thus, the expression of PgDDS in A. elata improved its medicinal value.

CsCuAO1 Associated with CsAMADH1 Confers Drought Tolerance by Modulating GABA Levels in Tea Plants.

Our previous study showed that COPPER-CONTAINING AMINE OXIDASE (CuAO) and AMINOALDEHYDE DEHYDROGENASE (AMADH) could regulate the accumulation of γ-aminobutyric acid (GABA) in tea through the polyamine degradation pathway. However, their biological function in drought tolerance has not been determined. In this study, Camellia sinensis (Cs) CsCuAO1 associated with CsAMADH1 conferred drought tolerance, which modulated GABA levels in tea plants. The results showed that exogenous GABA spraying effectively alleviated the drought-induced physical damage. Arabidopsis lines overexpressing CsCuAO1 and CsAMADH1 exhibited enhanced resistance to drought, which promoted the synthesis of GABA and putrescine by stimulating reactive oxygen species' scavenging capacity and stomatal movement. However, the suppression of CsCuAO1 or CsAMADH1 in tea plants resulted in increased sensitivity to drought treatment. Moreover, co-overexpressing plants increased GABA accumulation both in an Agrobacterium-mediated Nicotiana benthamiana transient assay and transgenic Arabidopsis plants. In addition, a GABA transporter gene, CsGAT1, was identified, whose expression was strongly correlated with GABA accumulation levels in different tissues under drought stress. Taken together, CsCuAO1 and CsAMADH1 were involved in the response to drought stress through a dynamic GABA-putrescine balance. Our data will contribute to the characterization of GABA's biological functions in response to environmental stresses in plants.

Genome-wide analysis of the U-box E3 ligases gene family in potato (Solanum tuberosum L.) and overexpress StPUB25 enhance drought tolerance in transgenic Arabidopsis.

BACKGROUND: Plant U-box (PUB) E3 ubiquitin ligases have vital effects on various biological processes. Therefore, a comprehensive and systematic identification of the members of the U-box gene family in potato will help to understand the evolution and function of U-box E3 ubiquitin ligases in plants. RESULTS: This work identified altogether 74 PUBs in the potato (StPUBs) and examined their gene structures, chromosomal distributions, and conserved motifs. There were seventy-four StPUB genes on ten chromosomes with diverse densities. As revealed by phylogenetic analysis on PUBs within potato, Arabidopsis, tomato (Solanum lycopersicum), cabbage (Brassica oleracea), rice (Oryza sativa), and corn (Zea mays), were clustered into eight subclasses (C1-C8). According to synteny analysis, there were 40 orthologous StPUB genes to Arabidopsis, 58 to tomato, 28 to cabbage, 7 to rice, and 8 to corn. In addition, RNA-seq data downloaded from PGSC were utilized to reveal StPUBs' abiotic stress responses and tissue-specific expression in the doubled-monoploid potato (DM). Inaddition, we performed RNA-seq on the 'Atlantic' (drought-sensitive cultivar, DS) and the 'Qingshu NO.9' (drought-tolerant cultivar, DT) in early flowering, full-blooming, along with flower-falling stages to detect genes that might be involved in response to drought stress. Finally, quantitative real-time PCR (qPCR) was carried out to analyze three candidate genes for their expression levels within 100 mM NaCl- and 10% PEG 6000 (w/v)-treated potato plantlets for a 24-h period. Furthermore, we analyzed the drought tolerance of StPUB25 transgenic plants and found that overexpression of StPUB25 significantly increased peroxidase (POD) activity, reduced ROS (reactive oxygen species) and MDA (malondialdehyde) accumulation compared with wild-type (WT) plants, and enhancing drought tolerance of the transgenic plants. CONCLUSION: In this study, three candidate genes related to drought tolerance in potato were excavated, and the function of StPUB25 under drought stress was verified. These results should provide valuable information to understand the potato StPUB gene family and investigate the molecular mechanisms of StPUBs regulating potato drought tolerance.

Transcriptome analysis of axillary buds in low phosphorus stress and functional analysis of TaWRKY74s in wheat.

BACKGROUND: Wheat is one of the main grain crops in the world, and the tiller number is a key factor affecting the yield of wheat. Phosphorus is an essential element for tiller development in wheat. However, due to decreasing phosphorus content in soil, there has been increasing use of phosphorus fertilizer, while imposing risk of soil and water pollution. Hence, it is important to identify low phosphorus tolerance genes and utilize them for stress resistance breeding in wheat. RESULTS: We subjected the wheat variety Kenong 199 (KN199) to low phosphorus stress and observed a reduced tiller number. Using transcriptome analysis, we identified 1651 upregulated genes and 827 downregulated of genes after low phosphorus stress. The differentially expressed genes were found to be enriched in the enzyme activity regulation related to phosphorus, hormone signal transduction, and ion transmembrane transport. Furthermore, the transcription factor analysis revealed that TaWRKY74s were important for low phosphorus tolerance. TaWRKY74s have three alleles: TaWRKY74-A, TaWRKY74-B, and TaWRKY74-D, and they all belong to the WRKY family with conserved WRKYGQK motifs. These proteins were found to be located in the nucleus, and they were expressed in axillary meristem, shoot apical meristem(SAM), young leaves, leaf primordium, and spikelet primordium. The evolutionary tree showed that TaWRKY74s were closely related to OsWRKY74s in rice. Moreover, TaWRKY74s-RNAi transgenic plants displayed significantly fewer tillers compared to wild-type plants under normal conditions. Additionally, the tiller numebr of the RNAi transgenic plants was also significantly lower than that of the wild-type plants under low-phosphorus stress, and increased the decrease amplitude. This suggestd that TaWRKY74s are related to phosphorus response and can affect the tiller number of wheat. CONCLUSIONS: The results of this research showed that TaWRKY74s were key genes in wheat response to low phosphorus stress, which might regulate wheat tiller number through abscisic acid (ABA) and auxin signal transduction pathways. This research lays the foundation for further investigating the mechanism of TaWRKY74s in the low phosphorus environments and is significant for wheat stress resistance breeding.

Simple method for transformation and gene editing in medicinal plants.

A sample delivery method, modified from cut-dip-budding, uses explants with robust shoot regeneration ability, enabling transformation and gene editing in medicinal plants, bypassing tissue culture and hairy root formation. This method has potential for applications across a wide range of plant species.

StBIN2 Positively Regulates Potato Formation through Hormone and Sugar Signaling.

Potato is an important food crop worldwide. Brassinosteroids (BRs) are widely involved in plant growth and development, and BIN2 (brassinosteroid insensitive 2) is the negative regulator of their signal transduction. However, the function of BIN2 in the formation of potato tubers remains unclear. In this study, transgenic methods were used to regulate the expression level of StBIN2 in plants, and tuber related phenotypes were analyzed. The overexpression of StBIN2 significantly increased the number of potatoes formed per plant and the weight of potatoes in transgenic plants. In order to further explore the effect of StBIN2 on the formation of potato tubers, this study analyzed BRs, ABA hormone signal transduction, sucrose starch synthase activity, the expression levels of related genes, and interacting proteins. The results show that the overexpression of StBIN2 enhanced the downstream transmission of ABA signals. At the same time, the enzyme activity of the sugar transporter and the expression of synthetic genes were increased in potato plants overexpressing StBIN2, which also demonstrated the upregulation of sucrose and the expression of the starch synthesis gene. Apparently, StBIN2 affected the conversion and utilization of key substances such as glucose, sucrose, and starch in the process of potato formation so as to provide a material basis and energy preparation for forming potatoes. In addition, StBIN2 also promoted the expression of the tuber formation factors StSP6A and StS6K. Altogether, this investigation enriches the study on the mechanism through which StBIN2 regulates potato tuber formation and provides a theoretical basis for achieving a high and stable yield of potato.

Genome-Wide Analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Family in Perilla frutescens and Functional Characterization of PfGPAT9 Crucial for Biosynthesis of Storage Oils Rich in High-Value Lipids.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol (TAG) biosynthesis. However, GPAT members and their functions remain poorly understood in Perilla frutescens, a special edible-medicinal plant with its seed oil rich in polyunsaturated fatty acids (mostly α-linolenic acid, ALA). Here, 14 PfGPATs were identified from the P. frutescens genome and classified into three distinct groups according to their phylogenetic relationships. These 14 PfGPAT genes were distributed unevenly across 11 chromosomes. PfGPAT members within the same subfamily had highly conserved gene structures and four signature functional domains, despite considerable variations detected in these conserved motifs between groups. RNA-seq and RT-qPCR combined with dynamic analysis of oil and FA profiles during seed development indicated that PfGPAT9 may play a crucial role in the biosynthesis and accumulation of seed oil and PUFAs. Ex vivo enzymatic assay using the yeast expression system evidenced that PfGPAT9 had a strong GPAT enzyme activity crucial for TAG assembly and also a high substrate preference for oleic acid (OA, C18:1) and ALA (C18:3). Heterogeneous expression of PfGPAT9 significantly increased total oil and UFA (mostly C18:1 and C18:3) levels in both the seeds and leaves of the transgenic tobacco plants. Moreover, these transgenic tobacco lines exhibited no significant negative effect on other agronomic traits, including plant growth and seed germination rate, as well as other morphological and developmental properties. Collectively, our findings provide important insights into understanding PfGPAT functions, demonstrating that PfGPAT9 is the desirable target in metabolic engineering for increasing storage oil enriched with valuable FA profiles in oilseed crops.

Compositional and Morphological Analysis of Salt Stress Tolerant Mannitol-1-phosphate Dehydrogenase (mtlD)-Transgenic Potato Plants.

Undesired effects often occur in genetically modified (GM) plants, especially during metabolite engineering. Nevertheless, conducting a comparative study between GM and non-GM plants can identify the unintended alterations and facilitate the risk assessment of GM crops. This research compared the morphology and composition of a transgenic potato plant expressing mannitol-1-phosphate dehydrogenase (mtlD), with its non-transgenic counterpart. The results indicated significant differences in plant height, number of leaves, length and width of leaves, as well as tuber number and weight between the transgenic and non-transgenic plants. However, compositional analysis revealed no significant differences in soluble protein, starch, total sugar, fructose, fiber, and ascorbate contents between mtlD-GM and non-GM potatoes. Nevertheless, sucrose and glucose levels were found to be higher in the transgenic potato tubers and leaves, respectively, when compared to the non-transgenic plants. In addition to ammonium, potassium, chloride, nitrite, and nitrate levels, significant differences were observed in the amino acids asparagine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, and valine between the GM and non-GM plants. Apart from the target gene product, mannitol, all the changes in chemical compositions observed in the transgenic potato plants fell within the ranges of normal variability for potato plants. Moreover, despite some phenotypical differences between the mtlD-GM potato and its non-GM counterpart, it is believed that this variation is a common phenomenon among potato varieties. In conclusion, the morphological and compositional analysis of the mtlD-GM potato plant revealed substantial equivalence with its non-transgenic counterpart.

Genetic manipulation of protein phosphatase 2A affects multiple agronomic traits and physiological parameters in potato (Solanum tuberosum).

In this study, agronomic and functional characteristics of potato (Solanum tuberosum ) plants constitutively overexpressing the protein phosphatase 2A (PP2A) catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were evaluated. StPP2Ac2b-OE plants display reduced vegetative growth, tuber yield and tuber weight under well-watered and drought conditions. Leaves of StPP2Ac2b-OE plants show an increased rate of water loss, associated with an impaired ability to close stomata in response to abscisic acid. StPP2Ac2b-OE lines exhibit larger stomatal size and reduced stomatal density. These altered stomatal characteristics might be responsible for the impaired stomatal closure and the elevated transpiration rates, ultimately leading to increased sensitivity to water-deficit stress and greater yield loss under drought conditions. Overexpression of StPP2Ac2b accelerates senescence in response to water-deficit stress, which could also contribute to the increased sensitivity to drought. Actively photosynthesising leaves of StPP2Ac2b-OE plants exhibit elevated levels of carbohydrates and a down-regulation of the sucrose transporter StSWEET11 , suggesting a reduced sucrose export from leaves to developing tubers. This effect, combined with the hindered vegetative development, may contribute to the reduced tuber weight and yield in StPP2Ac2b-OE plants. These findings offer novel insights into the physiological functions of PP2A in potato plants and provide valuable information for enhancing potato productivity by modulating the expression of StPP2Ac2b .

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Seed-specific expression of the class 2 Phytoglobin (Pgb2) increases seed oil in Brassica napus.

To examine the function of phytoglobin 2 (Pgb2) on seed oil level in the oil-producing crop Brassica napus L., we generated transgenic plants in which BnPgb2 was over-expressed in the seeds using the cruciferin1 promoter. Over-expression of BnPgb2 elevated the amount of oil, which showed a positive relationship with the level of BnPgb2, without altering the oil nutritional value, as evidenced by the lack of major changes in composition of fatty acids (FA), and key agronomic traits. Two key transcription factors, LEAFY COTYLEDON1 (LEC1) and WRINKLED1 (WRI1), known to promote the synthesis of fatty acids (FA) and potentiate oil accumulation, were induced in BnPgb2 over-expressing seeds. The concomitant induction of several enzymes of sucrose metabolism, SUCROSE SYNTHASE1 (SUS) 1 and 3, FRUCTOSE BISPHOSPHATE ALDOLASE (FPA), and PHOSPHOGLYCERATE KINASE (PGK), and starch synthesis, ADP-GLUCOSE PHOSPHORYLASE (AGPase) suggests that BnPgb2 favors sugar mobilization for FA production. The two plastid FA biosynthetic enzymes SUBUNIT A OF ACETYL-CoA CARBOXYLASE (ACCA2), and MALONYL-CoA:ACP TRANSACYLASE (MCAT) were also up-regulated by the over-expression of BnPgb2. The requirement of BnPgb2 for oil deposition was further evidenced in natural germplasm by the higher levels of BnPgb2 in seeds of high-oil genotypes relative to their low-oil counterparts.

Pre-Treatment of Rice Plants with ABA Makes Them More Tolerant to Multiple Abiotic Stress.

Multiple abiotic stress is known as a type of environmental unfavourable condition maximizing the yield and growth gap of crops compared with the optimal condition in both natural and cultivated environments. Rice is the world's most important staple food, and its production is limited the most by environmental unfavourable conditions. In this study, we investigated the pre-treatment of abscisic acid (ABA) on the tolerance of the IAC1131 rice genotype to multiple abiotic stress after a 4-day exposure to combined drought, salt and extreme temperature treatments. A total of 3285 proteins were identified and quantified across the four treatment groups, consisting of control and stressed plants with and without pre-treatment with ABA, with 1633 of those proteins found to be differentially abundant between groups. Compared with the control condition, pre-treatment with the ABA hormone significantly mitigated the leaf damage against combined abiotic stress at the proteome level. Furthermore, the application of exogenous ABA did not affect the proteome profile of the control plants remarkably, while the results were different in stress-exposed plants by a greater number of proteins changed in abundance, especially those which were increased. Taken together, these results suggest that exogenous ABA has a potential priming effect for enhancing the rice seedlings' tolerance against combined abiotic stress, mainly by affecting stress-responsive mechanisms dependent on ABA signalling pathways in plants.

Dioscorea composita WRKY5 positively regulates AtSOD1 and AtABF2 to enhance drought and salt tolerances.

KEY MESSAGE: DcWRKY5 increases the antioxidant enzyme activity and proline accumulation, oppositely, reduces the accumulation of ROS and MDA, through directly activating the genes expression, finally enhances the salt and drought tolerance. Drought and salinity are two main environmental factors that limit the large-scale cultivation of the medicinal plant Dioscorea composita (D. composita). WRKY transcription factors (TFs) play vital roles in regulating drought and salt tolerance in plants. Nevertheless, the molecular mechanism of WRKY TF mediates drought and salt resistance of D. composita remains largely unknown. Here, we isolated and characterized a WRKY TF from D. composita, namely DcWRKY5, which was localized to the nucleus and bound to the W-box cis-acting elements. Expression pattern analysis showed that it was highly expressed in root and significantly up-regulated in the presence of salt, polyethylene glycol-6000 (PEG-6000) and abscisic acid (ABA). Heterologous expression of DcWRKY5 increased salt and drought tolerance in Arabidopsis, but was insensitive to ABA. In addition, compared with the wild type, the DcWRKY5 overexpressing transgenic lines had more proline, higher antioxidant enzyme (POD, SOD, and CAT) activities, less reactive oxygen species (ROS) and malondialdehyde (MDA). Correspondingly, the overexpression of DcWRKY5 modulated the expression of genes related to salt and drought stresses, such as AtSS1, AtP5CS1, AtCAT, AtSOD1, AtRD22, and AtABF2. Dual luciferase assay and Y1H were further confirmed that DcWRKY5 activate the promoter of AtSOD1 and AtABF2 through directly binding to the enrichment region of the W-box cis-acting elements. These results suggest that DcWRKY5 is a positive regulator of the drought and salt tolerance in D. composita and has potential applications in transgenic breeding.

Establishment of an efficient transformation system and its application in regulatory mechanism analysis of biological macromolecules in tea plants.

Tea (Camellia sinensis), one of the most important beverage crops originated from China and is now cultivated worldwide, provides numerous secondary metabolites that account for its health benefits and rich flavor. However, the lack of an efficient and reliable genetic transformation system has seriously hindered the gene function investigation and precise breeding of C. sinensis. In this study, we established a highly efficient, labor-saving, and cost-effective Agrobacterium rhizogenes-mediated hairy roots genetic transformation system for C. sinensis, which can be used for gene overexpression and genome editing. The established transformation system was simple to operate, bypassing tissue culture and antibiotic screening, and only took two months to complete. We used this system to conduct function analysis of transcription factor CsMYB73 and found that CsMYB73 negatively regulates L-theanine synthesis in tea plant. Additionally, callus formation was successfully induced using transgenic roots, and the transgenic callus exhibited normal chlorophyll production, enabling the study of the corresponding biological functions. Furthermore, this genetic transformation system was effective for multiple C. sinensis varieties and other woody plant species. By overcoming technical obstacles such as low efficiency, long experimental periods, and high costs, this genetic transformation will be a valuable tool for routine gene investigation and precise breeding in tea plants.

Overexpression of potato ORANGE (StOR) and StOR mutant in Arabidopsis confers increased carotenoid accumulation and tolerance to abiotic stress.

ORANGE (OR) plays essential roles in regulating carotenoid homeostasis and enhancing the ability of plants to adapt to environmental stress. However, OR proteins have been functionally characterized in only a few plant species, and little is known about the role of potato OR (StOR). In this study, we characterized the StOR gene in potato (Solanum tuberosum L. cv. Atlantic). StOR is predominantly localized to the chloroplast, and its transcripts are tissue-specifically expressed and significantly induced in response to abiotic stress. Compared with wild type, overexpression of StOR increased ß-carotene levels up to 4.8-fold, whereas overexpression of StORHis with a conserved arginine to histidine substitution promoted ß-carotene accumulation up to 17.6-fold in Arabidopsis thaliana calli. Neither StOR nor StORHis overexpression dramatically affected the transcript levels of carotenoid biosynthetic genes. Furthermore, overexpression of either StOR or StORHis increased abiotic stress tolerance in Arabidopsis, which was associated with higher photosynthetic capacity and antioxidative activity. Taken together, these results indicate that StOR could be exploited as a potential new genetic tool for the improvement of crop nutritional quality and environmental stress tolerance.