HgS2, a novel salt-responsive gene from the Halophyte Halogeton glomeratus, confers salt tolerance in transgenic Arabidopsis.

Halophyte Halogeton glomeratus mostly grows in saline desert areas in arid and semi-arid regions and is able to adapt to adverse conditions such as salinity and drought. Earlier transcriptomic studies revealed activation of the HgS2 gene in the leaf of H. glomeratus seedlings when exposed to saline conditions. To identify the properties of HgS2 in H. glomeratus, we used yeast transformation and overexpression in Arabidopsis. Yeast cells genetically transformed with HgS2 exhibited K+ uptake and Na+ efflux compared with control (empty vector). Stable overexpression of HgS2 in Arabidopsis improved its resistance to salt stress and led to a notable rise in seed germination in salinity conditions compared to the wild type (WT). Transgenic Arabidopsis regulated ion homeostasis in plant cells by increasing Na+ absorption and decreasing K+ efflux in leaves, while reducing Na+ absorption and K+ efflux in roots. In addition, overexpression of HgS2 altered transcription levels of stress response genes and regulated different metabolic pathways in roots and leaves of Arabidopsis. These results offer new insights into the role of HgS2 in plants' salt tolerance.

Positive roles of the Ca2+ sensors GbCML45 and GbCML50 in improving cotton Verticillium wilt resistance.

As a universal second messenger, cytosolic calcium (Ca2+) functions in multifaceted intracellular processes, including growth, development and responses to biotic/abiotic stresses in plant. The plant-specific Ca2+ sensors, calmodulin and calmodulin-like (CML) proteins, function as members of the second-messenger system to transfer Ca2+ signal into downstream responses. However, the functions of CMLs in the responses of cotton (Gossypium spp.) after Verticillium dahliae infection, which causes the serious vascular disease Verticillium wilt, remain elusive. Here, we discovered that the expression level of GbCML45 was promoted after V. dahliae infection in roots of cotton, suggesting its potential role in Verticillium wilt resistance. We found that knockdown of GbCML45 in cotton plants decreased resistance while overexpression of GbCML45 in Arabidopsis thaliana plants enhanced resistance to V. dahliae infection. Furthermore, there was physiological interaction between GbCML45 and its close homologue GbCML50 by using yeast two-hybrid and bimolecular fluorescence assays, and both proteins enhanced cotton resistance to V. dahliae infection in a Ca2+-dependent way in a knockdown study. Detailed investigations indicated that several defence-related pathways, including salicylic acid, ethylene, reactive oxygen species and nitric oxide signalling pathways, as well as accumulations of lignin and callose, are responsible for GbCML45- and GbCML50-modulated V. dahliae resistance in cotton. These results collectively indicated that GbCML45 and GbCML50 act as positive regulators to improve cotton Verticillium wilt resistance, providing potential targets for exploitation of improved Verticillium wilt-tolerant cotton cultivars by genetic engineering and molecular breeding.

Sugarcane ScOPR1 gene enhances plant disease resistance through the modulation of hormonal signaling pathways.

KEY MESSAGE: Transgenic plants stably overexpressing ScOPR1 gene enhanced disease resistance by increasing the accumulation of JA, SA, and GST, as well as up-regulating the expression of genes related to signaling pathways. 12-Oxo-phytodienoate reductase (OPR) is an oxidoreductase that depends on flavin mononucleotide (FMN) and catalyzes the conversion of 12-oxophytodienoate (12-OPDA) into jasmonic acid (JA). It plays a key role in plant growth and development, and resistance to adverse stresses. In our previous study, we have obtained an OPR gene (ScOPR1, GenBank Accession Number: MG755745) from sugarcane. This gene showed positive responses to methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), and Sporisorium scitamineum, suggesting its potential for pathogen resistance. Here, in our study, we observed that Nicotiana benthamiana leaves transiently overexpressing ScOPR1 exhibited weaker disease symptoms, darker 3,3-diaminobenzidine (DAB) staining, higher accumulation of reactive oxygen species (ROS), and higher expression of hypersensitive response (HR) and SA pathway-related genes after inoculation with Ralstonia solanacearum and Fusarium solanacearum var. coeruleum. Furthermore, the transgenic N. benthamiana plants stably overexpressing the ScOPR1 gene showed enhanced resistance to pathogen infection by increasing the accumulation of JA, SA, and glutathione S-transferase (GST), as well as up-regulating genes related to HR, JA, SA, and ROS signaling pathways. Transcriptome analysis revealed that the specific differentially expressed genes (DEGs) in ScOPR1-OE were significantly enriched in hormone transduction signaling and plant-pathogen interaction pathways. Finally, a functional mechanism model of the ScOPR1 gene in response to pathogen infection was depicted. This study provides insights into the molecular mechanism of ScOPR1 and presents compelling evidence supporting its positive involvement in enhancing plant disease resistance.

AcEXPA1, an α-expansin gene, participates in the aluminum tolerance of carpetgrass (Axonopus compressus) through root growth regulation.

KEY MESSAGE: AcEXPA1, an aluminum (Al)-inducible expansin gene, is demonstrated to be involved in carpetgrass (Axonopus compressus) root elongation under Al toxicity through analyzing composite carpetgrass plants overexpressing AcEXPA1. Aluminum (Al) toxicity is a major mineral toxicity that limits plant productivity in acidic soils by inhibiting root growth. Carpetgrass (Axonopus compressus), a dominant warm-season turfgrass widely grown in acidic tropical soils, exhibits superior adaptability to Al toxicity. However, the mechanisms underlying its Al tolerance are largely unclear, and knowledge of the functional genes involved in Al detoxification in this turfgrass is limited. In this study, phenotypic variation in Al tolerance, as indicated by relative root elongation, was observed among seventeen carpetgrass genotypes. Al-responsive genes related to cell wall modification were identified in the roots of the Al-tolerant genotype 'A58' via transcriptome analysis. Among them, a gene encoding α-expansin was cloned and designated AcEXPA1 for functional characterization. Observed Al dose effects and temporal responses revealed that Al induced AcEXPA1 expression in carpetgrass roots. Subsequently, an efficient and convenient Agrobacterium rhizogenes-mediated transformation method was established to generate composite carpetgrass plants with transgenic hairy roots for investigating AcEXPA1 involvement in carpetgrass root growth under Al toxicity. AcEXPA1 was successfully overexpressed in the transgenic hairy roots, and AcEXPA1 overexpression enhanced Al tolerance in composite carpetgrass plants through a decrease in Al-induced root growth inhibition. Taken together, these findings suggest that AcEXPA1 contributes to Al tolerance in carpetgrass via root growth regulation.

In vivo transgenic studies confirm the critical acylation function of LeBAHD56 for shikonin in Lithospermum erythrorhizon.

KEY MESSAGE: LeBAHD56 is preferentially expressed in tissues where shikonin and its derivatives are biosynthesized, and it confers shikonin acylation in vivo. Two WRKY transcriptional factors might regulate LeBAHD56's expression. Shikonin and its derivatives, found in the roots of Lithospermum erythrorhizon, have extensive application in the field of medicine, cosmetics, and other industries. Prior research has demonstrated that LeBAHD1(LeSAT1) is responsible for the biochemical process of shikonin acylation both in vitro and in vivo. However, with the exception of its documented in vitro biochemical function, there is no in vivo genetic evidence supporting the acylation function of the highly homologous gene of LeSAT1, LeBAHD56(LeSAT2), apart from its reported role. Here, we validated the critical acylation function of LeBAHD56 for shikonin using overexpression (OE) and CRISPR/Cas9-based knockout (KO) strategies. The results showed that the OE lines had a significantly higher ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than the control. In contrast, the KO lines had a significantly lower ratio of acetylshikonin, isobutyrylshikonin or isovalerylshikonin to shikonin than controls. As for its detailed expression patterns, we found that LeBAHD56 is preferentially expressed in roots and callus cells, which are the biosynthesis sites for shikonin and its derivatives. In addition, we anticipated that a wide range of putative transcription factors might control its transcription and verified the direct binding of two crucial WRKY members to the LeBAHD56 promoter's W-box. Our results not only confirmed the in vivo function of LeBAHD56 in shikonin acylation, but also shed light on its transcriptional regulation.

Minimizing IP issues associated with gene constructs encoding the Bt toxin - a case study.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants. RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves. CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

The 14-3-3 protein nt GF14e interacts with CIPK2 and increases low potassium stress in tobacco.

Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.

Effect of SlSAHH2 on metabolites in over-expressed and wild-type tomato fruit.

Background: Tomato (Solanum lycopersicum) is an annual or perennial herb that occupies an important position in daily agricultural production. It is an essential food crop for humans and its ripening process is regulated by a number of genes. S-adenosyl-l-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) is widespread in organisms and plays an important role in regulating biological methylation reactions. Previous studies have revealed that transgenic tomato that over-express SlSAHH2 ripen earlier than the wild-type (WT). However, the differences in metabolites and the mechanisms driving how these differences affect the ripening cycle are unclear. Objective: To investigate the effects of SlSAHH2 on metabolites in over-expressed tomato and WT tomato. Methods: SlSAHH2 over-expressed tomato fruit (OE-5# and OE-6#) and WT tomato fruit at the breaker stage (Br) were selected for non-targeted metabolome analysis. Results: A total of 733 metabolites were identified by mass spectrometry using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Human Metabolome database (HMDB). The metabolites were divided into 12 categories based on the superclass results and a comparison with the HMDB. The differences between the two databases were analyzed by PLS-DA. Based on a variable important in projection value >1 and P < 0.05, 103 differential metabolites were found between tomato variety OE-5# and WT and 63 differential metabolites were found between OE-6# and WT. These included dehydrotomatine, L-serine, and gallic acid amongst others. Many metabolites are associated with fruit ripening and eight common metabolites were found between the OE-5# vs. WT and OE-6# vs. WT comparison groups. The low L-tryptophan expression in OE-5# and OE-6# is consistent with previous reports that its content decreases with fruit ripening. A KEGG pathway enrichment analysis of the significantly different metabolites revealed that in the OE-5# and WT groups, up-regulated metabolites were enriched in 23 metabolic pathways and down-regulated metabolites were enriched in 11 metabolic pathways. In the OE-6# and WT groups, up-regulated metabolites were enriched in 29 pathways and down-regulated metabolites were enriched in six metabolic pathways. In addition, the differential metabolite changes in the L-serine to flavonoid transformation metabolic pathway also provide evidence that there is a phenotypic explanation for the changes in transgenic tomato. Discussion: The metabolomic mechanism controlling SlSAHH2 promotion of tomato fruit ripening has been further elucidated.

Efficient tobacco rattle virus-induced gene editing in tomato mediated by the CRISPR/Cas9 system.

Plant virus-based sgRNA delivery strategy has been widely applied for efficient genome editing across various plant species, leveraging its significant advantages in the rapid expression and expansion of sgRNA through virus replication and movement. However, the efficacy of the virus-induced gene editing (VIGE) tool in tomato has yet to be explored. In this paper, we established a TRV-mediated CRISPR/Cas9 genome editing system in the somatic cells of tomato, reporting the validation of VIGE and evaluating the mutagenesis efficiency in both tomato leaves and fruits using high-throughput sequencing. The results demonstrated an approximate 65% efficiency of VIGE in tomato leaves for the selected target genes, with VIGE efficiency reaching up to 50% in tomato fruits. This research not only introduces an efficient tool for reverse genetics but also reveals substantial potential of VIGE in surpassing traditional tissue culture techniques for creating heritable mutations in tomato.

Development and application of an RNA nanostructure to induce transient RNAi in difficult transgenic plants.

The development of RNA interference (RNAi) is crucial for studying plant gene function. Its use, is limited to a few plants with well-established transgenic techniques. Spray-induced gene silencing (SIGS) introduces exogenous double-stranded RNA (dsRNA) into plants by spraying, injection, or irrigation, triggering the RNAi pathway to instantly silence target genes. As is a transient RNAi technology that does not rely on transgenic methods, SIGS has significant potential for studying gene function in plants lacking advanced transgenic technology. In this study, to enhance their stability and delivery efficiency, siRNAs were used as structural motifs to construct RNA nanoparticles (NPs) of four shapes: triangle, square, pentagon, and hexagon. These NPs, when synthesized by Escherichia coli, showed that triangular and square shapes accumulated more efficiently than pentagon and hexagon shapes. Bioassays revealed that RNA squares had the highest RNAi efficiency, followed by RNA triangles, with GFP-dsRNA showing the lowest efficiency at 4 and 7 days post-spray. We further explored the use of RNA squares in inducing transient RNAi in plants that are difficult to transform genetically. The results indicated that Panax notoginseng-derived MYB2 (PnMYB2) and Camellia oleifera-derived GUT (CoGUT) were significantly suppressed in P. notoginseng and C. oleifera, respectively, following the application of PnMYB2- and CoGUT-specific RNA squares. These findings suggest that RNA squares are highly effective in SIGS and can be utilized for gene function research in plants.

Mitigation of UV-B Radiation Stress in Tobacco Pollen by Expression of the Tardigrade Damage Suppressor Protein (Dsup).

Pollen, the male gametophyte of seed plants, is extremely sensitive to UV light, which may prevent fertilization. As a result, strategies to improve plant resistance to solar ultraviolet (UV) radiation are required. The tardigrade damage suppressor protein (Dsup) is a putative DNA-binding protein that enables tardigrades to tolerate harsh environmental conditions, including UV radiation, and was therefore considered as a candidate for reducing the effects of UV exposure on pollen. Tobacco pollen was genetically engineered to express Dsup and then exposed to UV-B radiation to determine the effectiveness of the protein in increasing pollen resistance. To establish the preventive role of Dsup against UV-B stress, we carried out extensive investigations into pollen viability, germination rate, pollen tube length, male germ unit position, callose plug development, marker protein content, and antioxidant capacity. The results indicated that UV-B stress has a significant negative impact on both pollen grain and pollen tube growth. However, Dsup expression increased the antioxidant levels and reversed some of the UV-B-induced changes to pollen, restoring the proper distance between the tip and the last callose plug formed, as well as pollen tube length, tubulin, and HSP70 levels. Therefore, the expression of heterologous Dsup in pollen may provide the plant male gametophyte with enhanced responses to UV-B stress and protection against harmful environmental radiation.

Transcriptome and metabolome analyses reveal the regulatory role of MdPYL9 in drought resistance in apple.

BACKGROUND: The mechanisms by which the apple MdPYL9 gene mediates the response to drought stress remain unclear. Here, transcriptome and metabolome analyses of apple plants under drought were used to investigate the mechanisms by which MdPYL9 regulates the response to drought stress in apple. MdPYL9-overexpressed transgenic and non-transgenic apple histoculture seedlings were rooted, transplanted, and subjected to drought treatments to clarify the mechanisms underlying the responses of apples to drought stress through phenotypic observations, physiological and biochemical index measurements, and transcriptomic and metabolomic analyses. RESULTS: Under drought stress treatment, transgenic plants were less affected by drought stress than non-transgenic plants. Decreases in the net photosynthetic rate, stomatal conductance, and transpiration rate of transgenic apple plants were less pronounced in transgenic plants than in non-transgenic plants, and increases in the intercellular CO2 concentration were less pronounced in transgenic plants than in non-transgenic plants. The relative electrical conductivity and content of malondialdehyde, superoxide anion, and hydrogen peroxide were significantly lower in transgenic plants than in non-transgenic plants, and the chlorophyll content and activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) were significantly higher in transgenic plants than in non-transgenic plants. The number of differentially expressed genes (DEGs) involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and the most significant and highly annotated DEGs in the transgenic plants were involved in the flavonoid biosynthesis pathway, and the most significant and highly annotated DEGs in control plants were involved in the phytohormone signal transduction pathway. The number of differentially accumulated metabolites involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and up-regulated metabolites were significantly enriched in apigenin-7-O-glucoside in transgenic plants and in abscisic acid in non-transgenic plants. In the flavonoid biosynthetic pathway, the expression of genes encoding chalcone synthase (CHS) and chalcone isomerase (CHI) was more significantly down-regulated in non-transgenic plants than in transgenic plants, and the expression of the gene encoding 4-coumarate-CoA ligase (4CL) was more significantly up-regulated in transgenic plants than in non-transgenic plants, which resulted in the significant up-regulation of apigenin-7-O-glucoside in transgenic plants. CONCLUSIONS: The above results indicated that the over-expression of MdPYL9 increased the drought resistance of plants under drought stress by attenuating the down-regulation of the expression of genes encoding CHS and CHI and enhancing the up-regulated expression of the gene encoding 4CL, which enhanced the content of apigenin-7-O-glucoside.

Co-overexpression of chitinase and ß-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium.

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and ß-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and ß-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 µg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

GhCLCc-1, a Chloride Channel Gene from Upland Cotton, Positively Regulates Salt Tolerance by Modulating the Accumulation of Chloride Ions.

The ionic toxicity induced by salinization has adverse effects on the growth and development of crops. However, researches on ionic toxicity and salt tolerance in plants have focused primarily on cations such as sodium ions (Na+), with very limited studies on chloride ions (Cl-). Here, we cloned the homologous genes of Arabidopsis thaliana AtCLCc, GhCLCc-1A/D, from upland cotton (Gossypium hirsutum), which were significantly induced by NaCl or KCl treatments. Subcellular localization showed that GhCLCc-1A/D were both localized to the tonoplast. Complementation of Arabidopsis atclcc mutant with GhCLCc-1 rescued its salt-sensitive phenotype. In addition, the silencing of the GhCLCc-1 gene led to an increased accumulation of Cl- in the roots, stems, and leaves of cotton seedlings under salt treatments, resulting in compromised salt tolerance. And ectopic expression of the GhCLCc-1 gene in Arabidopsis reduced the accumulation of Cl- in transgenic lines under salt treatments, thereby enhancing salt tolerance. These findings elucidate that GhCLCc-1 positively regulates salt tolerance by modulating Cl- accumulation and could be a potential target gene for improving salt tolerance in plants.

Cloning and Function Analysis of the CsTAU1 in Response to Salt-Alkali Stress.

To investigate the role of candidate genes for salt-alkali tolerance in cucumber (Cucumis sativus L.), this study screened CsTAU1 in the glutathione pathway from previous transcriptome data for cloning and functional analysis. Clone cucumber CsTAU1 contains one 675 bp open reading frame, containing one GST-N-Tau domain and one GST-C-Tau domain, and is expressed in cytoplasm. After successfully constructing overexpression vectors of CsTAU1 (+) and CsTAU1 (-), they were transferred into cucumber varieties 'D1909' (high salt alkali resistance) and 'D1604' (low salt alkali resistance) for salt-alkali resistance identification. It was found that under salt-alkali stress, CsTAU1 (+)-overexpressing plants showed strong resistance to salt-alkali stress, while CsTAU1 (-)-overexpressing plants showed the opposite situation. qRT-PCR analysis was performed on other glutathione pathway-related genes in CsTAU1-overexpressing plants. The expression patterns of LOC101219529 and LOC105434443 were the same as CsTAU1, and the introduction of CsTAU1 (+) increased the chlorophyll, α-Naphthylamine oxidation, glutathione S-transferase (GST), and catalase (CAT) content of cucumber. The research results provide a theoretical basis for cultivating salt-alkali-tolerant cucumber varieties.

Genome-Wide Analysis of C/S1-bZIP Subfamilies in Populus tomentosa and Unraveling the Role of PtobZIP55/21 in Response to Low Energy.

C/S1 basic leucine zipper (bZIP) transcription factors are essential for plant survival under energy deficiency. However, studies on the responses of C/S1-bZIPs to low energy in woody plants have not yet been reported. In this study, members of C/S1-bZIP subfamilies in Populus tomentosa were systematically analyzed using bioinformatic approaches. Four C-bZIPs and 10 S1-bZIPs were identified, and their protein properties, phylogenetic relationships, gene structures, conserved motifs, and uORFs were systematically investigated. In yeast two-hybrid assays, direct physical interactions between C-bZIP and S1-bZIP members were observed, highlighting their potential functional synergy. Moreover, expression profile analyses revealed that low energy induced transcription levels of most C/S1-bZIP members, with bZIP55 and bZIP21 (a homolog of bZIP55) exhibiting particularly significant upregulation. When the expression of bZIP55 and bZIP21 was co-suppressed using artificial microRNA mediated gene silencing in transgenic poplars, root growth was promoted. Further analyses revealed that bZIP55/21 negatively regulated the root development of P. tomentosa in response to low energy. These findings provide insights into the molecular mechanisms by which C/S1-bZIPs regulate poplar growth and development in response to energy deprivation.

The RNA-Binding Protein BoRHON1 Positively Regulates the Accumulation of Aliphatic Glucosinolates in Cabbage.

Aliphatic glucosinolates are an abundant group of plant secondary metabolites in Brassica vegetables, with some of their degradation products demonstrating significant anti-cancer effects. The transcription factors MYB28 and MYB29 play key roles in the transcriptional regulation of aliphatic glucosinolates biosynthesis, but little is known about whether BoMYB28 and BoMYB29 are also modulated by upstream regulators or how, nor their gene regulatory networks. In this study, we first explored the hierarchical transcriptional regulatory networks of MYB28 and MYB29 in a model plant, then systemically screened the regulators of the three BoMYB28 homologs in cabbage using a yeast one-hybrid. Furthermore, we selected a novel RNA binding protein, BoRHON1, to functionally validate its roles in modulating aliphatic glucosinolates biosynthesis. Importantly, BoRHON1 induced the accumulation of all detectable aliphatic and indolic glucosinolates, and the net photosynthetic rates of BoRHON1 overexpression lines were significantly increased. Interestingly, the growth and biomass of these overexpression lines of BoRHON1 remained the same as those of the control plants. BoRHON1 was shown to be a novel, potent, positive regulator of glucosinolates biosynthesis, as well as a novel regulator of normal plant growth and development, while significantly increasing plants' defense costs.

Amaranth's Growth and Physiological Responses to Salt Stress and the Functional Analysis of AtrTCP1 Gene.

Amaranth species are C4 plants that are rich in betalains, and they are tolerant to salinity stress. A small family of plant-specific TCP transcription factors are involved in the response to salt stress. However, it has not been investigated whether amaranth TCP1 is involved in salt stress. We elucidated that the growth and physiology of amaranth were affected by salt concentrations of 50-200 mmol·L-1 NaCl. The data showed that shoot and root growth was inhibited at 200 mmol·L-1, while it was promoted at 50 mmol·L-1. Meanwhile, the plants also showed physiological responses, which indicated salt-induced injuries and adaptation to the salt stress. Moreover, AtrTCP1 promoted Arabidopsis seed germination. The germination rate of wild-type (WT) and 35S::AtrTCP1-GUS Arabidopsis seeds reached around 92% by the seventh day and 94.5% by the second day under normal conditions, respectively. With 150 mmol·L-1 NaCl treatment, the germination rate of the WT and 35S::AtrTCP1-GUS plant seeds was 27.0% by the seventh day and 93.0% by the fourth day, respectively. Under salt stress, the transformed 35S::AtrTCP1 plants bloomed when they grew 21.8 leaves after 16.2 days of treatment, which was earlier than the WT plants. The transformed Arabidopsis plants flowered early to resist salt stress. These results reveal amaranth's growth and physiological responses to salt stress, and provide valuable information on the AtrTCP1 gene.

A quantitative gibberellin signaling biosensor reveals a role for gibberellins in internode specification at the shoot apical meristem.

Growth at the shoot apical meristem (SAM) is essential for shoot architecture construction. The phytohormones gibberellins (GA) play a pivotal role in coordinating plant growth, but their role in the SAM remains mostly unknown. Here, we developed a ratiometric GA signaling biosensor by engineering one of the DELLA proteins, to suppress its master regulatory function in GA transcriptional responses while preserving its degradation upon GA sensing. We demonstrate that this degradation-based biosensor accurately reports on cellular changes in GA levels and perception during development. We used this biosensor to map GA signaling activity in the SAM. We show that high GA signaling is found primarily in cells located between organ primordia that are the precursors of internodes. By gain- and loss-of-function approaches, we further demonstrate that GAs regulate cell division plane orientation to establish the typical cellular organization of internodes, thus contributing to internode specification in the SAM.

Carotenoid biosynthesis genes LcLCYB, LcLCYE, and LcBCH from wolfberry confer increased carotenoid content and improved salt tolerance in tobacco.

Carotenoids play essential roles in plant growth and development and provide plants with a tolerance to a series of abiotic stresses. In this study, the function and biological significance of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase, which are responsible for the modification of the tetraterpene skeleton procedure, were isolated from Lycium chinense and analyzed. The overexpression of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase promoted the accumulation of total carotenoids and photosynthesis enhancement, reactive oxygen species scavenging activity, and proline content of tobacco seedlings after exposure to the salt stress. Furthermore, the expression of the carotenoid biosynthesis genes and stress-related genes (ascorbate peroxidase, catalase, peroxidase, superoxide dismutase, and pyrroline-5-carboxylate reductase) were detected and showed increased gene expression level, which were strongly associated with the carotenoid content and reactive oxygen species scavenging activity. After exposure to salt stress, the endogenous abscisic acid content was significantly increased and much higher than those in control plants. This research contributes to the development of new breeding aimed at obtaining stronger salt tolerance plants with increased total carotenoids and vitamin A content.