Promoter of Cassava MeAHL31 Responds to Diverse Abiotic Stresses and Hormone Signals in Transgenic Arabidopsis.

The AT-hook motif nuclear-localized (AHL) family is pivotal for the abiotic stress response in plants. However, the function of the cassava AHL genes has not been elucidated. Promoters, as important regulatory elements of gene expression, play a crucial role in stress resistance. In this study, the promoter of the cassava MeAHL31 gene was cloned. The MeAHL31 protein was localized to the cytoplasm and the nucleus. qRT-PCR analysis revealed that the MeAHL31 gene was expressed in almost all tissues tested, and the expression in tuber roots was 321.3 times higher than that in petioles. Promoter analysis showed that the MeAHL31 promoter contains drought, methyl jasmonate (MeJA), abscisic acid (ABA), and gibberellin (GA) cis-acting elements. Expression analysis indicated that the MeAHL31 gene is dramatically affected by treatments with salt, drought, MeJA, ABA, and GA3. Histochemical staining in the proMeAHL31-GUS transgenic Arabidopsis corroborated that the GUS staining was found in most tissues and organs, excluding seeds. Beta-glucuronidase (GUS) activity assays showed that the activities in the proMeAHL31-GUS transgenic Arabidopsis were enhanced by different concentrations of NaCl, mannitol (for simulating drought), and MeJA treatments. The integrated findings suggest that the MeAHL31 promoter responds to the abiotic stresses of salt and drought, and its activity is regulated by the MeJA hormone signal.

A teosinte-derived allele of ZmSC improves salt tolerance in maize.

Maize, a salt-sensitive crop, frequently suffers severe yield losses due to soil salinization. Enhancing salt tolerance in maize is crucial for maintaining yield stability. To address this, we developed an introgression line (IL76) through introgressive hybridization between maize wild relatives Zea perennis, Tripsacum dactyloides, and inbred Zheng58, utilizing the tri-species hybrid MTP as a genetic bridge. Previously, genetic variation analysis identified a polymorphic marker on Zm00001eb244520 (designated as ZmSC), which encodes a vesicle-sorting protein described as a salt-tolerant protein in the NCBI database. To characterize the identified polymorphic marker, we employed gene cloning and homologous cloning techniques. Gene cloning analysis revealed a non-synonymous mutation at the 1847th base of ZmSCIL76 , where a guanine-to-cytosine substitution resulted in the mutation of serine to threonine at the 119th amino acid sequence (using ZmSCZ58 as the reference sequence). Moreover, homologous cloning demonstrated that the variation site derived from Z. perennis. Functional analyses showed that transgenic Arabidopsis lines overexpressing ZmSCZ58 exhibited significant reductions in leaf number, root length, and pod number, alongside suppression of the expression of genes in the SOS and CDPK pathways associated with Ca2+ signaling. Similarly, fission yeast strains expressing ZmSCZ58 displayed inhibited growth. In contrast, the ZmSCIL76 allele from Z. perennis alleviated these negative effects in both Arabidopsis and yeast, with the lines overexpressing ZmSCIL76 exhibiting significantly higher abscisic acid (ABA) content compared to those overexpressing ZmSCZ58 . Our findings suggest that ZmSC negatively regulates salt tolerance in maize by suppressing downstream gene expression associated with Ca2+ signaling in the CDPK and SOS pathways. The ZmSCIL76 allele from Z. perennis, however, can mitigate this negative regulatory effect. These results provide valuable insights and genetic resources for future maize salt tolerance breeding programs.

Comparative Transcriptome Analysis Revealed Candidate Gene Modules Involved in Salt Stress Response in Sweet Basil and Overexpression of ObWRKY16 and ObPAL2 Enhanced Salt Tolerance of Transgenic Arabidopsis.

Sweet basil (Ocimum basilicum L.) is an important aromatic plant with high edibility and economic value, widely distributed in many regions of the tropics including the south of China. In recent years, environmental problems, especially soil salinization, have seriously restricted the planting and spread of sweet basil. However, the molecular mechanism of the salt stress response in sweet basil is still largely unknown. In this study, seed germination, seedling growth, and chlorophyll synthesis in sweet basil were inhibited under salt stress conditions. Through comparative transcriptome analysis, the gene modules involved in the metabolic processes, oxidative response, phytohormone signaling, cytoskeleton, and photosynthesis were screened out. In addition, the landscape of transcription factors during salt treatment in sweet basil was displayed as well. Moreover, the overexpression of the WRKY transcription factor-encoding gene, ObWRKY16, and the phenylalanine ammonia-lyase-encoding gene, ObPAL2, enhanced the seed germination, seedling growth, and survival rate, respectively, of transgenic Arabidopsis, suggesting that they might be important candidates for the creation of salt-tolerant sweet basil cultivars. Our data enrich the study on salt responses in sweet basil and provide essential gene resources for genetic improvements in sweet basil in the future.

A cyclic nucleotide-gated channel gene HcCNGC21 positively regulates salt and drought stress responses in kenaf (Hibiscus cannabinus L.).

Cyclic Nucleotide-Gated Channels (CNGCs) serve as Ca2+ permeable cation transport pathways, which are involved in the regulation of various biological functions such as plant cell ion selective permeability, growth and development, responses to biotic and abiotic stresses. At the present study, a total of 31 CNGC genes were identified and bioinformatically analyzed in kenaf. Among these genes, HcCNGC21 characterized to localize at the plasma membrane, with the highest expression levels in leaves, followed by roots. In addition, HcCNGC21 could be significantly induced under salt or drought stress. Virus-induced gene silencing (VIGS) of HcCNGC21 in kenaf caused notable growth inhibition under salt or drought stress, characterized by reductions in plant height, stem diameter, leaf area, root length, root surface area, and root tip number. Meanwhile, the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly decreased, accompanied by reduced levels of osmoregulatory substances and total chlorophyll content. However, ROS accumulation and Na+ content increased. The expression of stress-responsive genes, such as HcSOD, HcPOD, HcCAT, HcERF3, HcNAC29, HcP5CS, HcLTP, and HcNCED, was significantly downregulated in these silenced lines. However, under salt or drought stress, the physiological performance and expression of stress-related genes in transgenic Arabidopsis thaliana plants overexpressing HcCNGC21 were diametrically opposite to those of TRV2-HcCNGC21 kenaf line. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays revealed that HcCNGC21 interacts with HcAnnexin D1. These findings collectively underscore the positive role of HcCNGC21 in plant resistance to salt and drought stress.

Over-expression of Medicago Acyl-CoA-binding 2 genes enhance salt and drought tolerance in Arabidopsis.

Acyl-CoA-binding proteins (ACBPs) are mainly involved in acyl-CoA ester binding and trafficking in eukaryotic cells, and they function in lipid metabolism, membrane biosynthesis, cellular signaling, stress response, disease resistance, and other biological activities in plants. However, the roles of ACBP family members in Medicago remain unclear. In this study, a total of eight ACBP genes were identified in the genome of Medicago truncatula and Medicago sativa, and they were clustered into four sub-families (Class I-IV). Many cis-acting elements related to abiotic response were identified in the promoter region of these ACBP genes, in particular light-responsive elements. These ACBP genes exhibited distinct expression pattern in various tissues, and the expression level of MtACBP1/MsACBP1 and MtACBP2/MsACBP2 gene pairs were significantly increased under NaCl treatment. Subcellular localization analysis showed that MtACBP1/MsACBP1 and MtACBP2/MsACBP2 were localized in the endoplasmic reticulum of tobacco epidermal cells. Arabidopsis seedlings over-expressing MtACBP2/MsACBP2 displayed increased root length than the wild type under short light, Cu2+, ABA, PEG, and NaCl treatments. Over-expression of MtACBP2/MsACBP2 also significantly enhanced Arabidopsis tolerance under NaCl and PEG treatments in mature plants. Collectively, our study identified salt and drought responsive ACBP genes in Medicago and verified their functions in increasing resistance against salt and drought stresses.

Preserving the adaptive salt stress response activity of a tissue-specific promoter with modulating activity.

BACKGROUND: The Arabidopsis "Redox Responsive Transcription Factor1" (RRTF1) promoter is transiently activated by salt stress in roots over 6 h period, followed by an adaptation phase during which its activity returns to baseline levels, even if the salt stress is prolonged. This enables the short-term production of genes that, while initially advantageous to the plant, will have long-term detrimental effects if expressed at high levels indefinitely. RESULTS: In this paper, we demonstrate that the RRTF1 promoter salt adaption response is a dominant feature of the promoter, that cannot be overwritten by a strong enhancer. While maintaining the transient activation profile of the RRTF1 promoter, linking it to the 35S enhancer results in a significant boost of salt stress induction in roots. CONCLUSION: The RRTF1 promoter's enhanced and still adaptable activity could become a useful tool in plant biotechnology.

Identification of ARF genes in Cucurbita pepo L and analysis of expression patterns, and functional analysis of CpARF22 under drought, salt stress.

BACKGROUND: Auxin transcription factor (ARF) is an important transcription factor that transmits auxin signals and is involved in plant growth and development as well as stress response. However, genome-wide identification and responses to abiotic and pathogen stresses of the ARF gene family in Cucurbita pepo L, especially pathogen stresses, have not been reported. RESULTS: Finally, 33 ARF genes (CpARF01 to CpARF33) were identified in C.pepo from the Cucurbitaceae genome database using bioinformatics methods. The putative protein contains 438 to 1071 amino acids, the isoelectric point is 4.99 to 8.54, and the molecular weight is 47759.36 to 117813.27 Da, the instability index ranged from 40.74 to 68.94, and the liposoluble index ranged from 62.56 to 76.18. The 33 genes were mainly localized in the nucleus and cytoplasm, and distributed on 16 chromosomes unevenly. Phylogenetic analysis showed that 33 CpARF proteins were divided into 6 groups. According to the amino acid sequence of CpARF proteins, 10 motifs were identified, and 1,3,6,8,10 motifs were highly conserved in most of the CpARF proteins. At the same time, it was found that genes in the same subfamily have similar gene structures. Cis-elements and protein interaction networks predicted that CpARF may be involved in abiotic factors related to the stress response. QRT-PCR analysis showed that most of the CpARF genes were upregulated under NaCl, PEG, and pathogen treatment compared to the control. Subcellular localization showed that CpARF22 was localized in the nucleus. The transgenic Arabidopsis thaliana lines with the CpARF22 gene enhanced their tolerance to salt and drought stress. CONCLUSION: In this study, we systematically analyzed the CpARF gene family and its expression patterns under drought, salt, and pathogen stress, which improved our understanding of the ARF protein of zucchini, and laid a solid foundation for functional analysis of the CpARF gene.

The thioredoxin h-type TdTrxh2 protein of durum wheat confers abiotic stress tolerance of the transformant Arabidopsis plants through its protective role and the regulation of redox homoeostasis.

The thioredoxins (Trxs) are ubiquitous and they play a crucial role in various biological processes like growth and stress response. Although the functions of Trxs proteins are described in several previous reports, the function of the isoform Trxh2 of durum wheat (Triticum durum L.), designated as TdTrxh2, in abiotic stress response still unknown. Thus, we aimed in this study the functional characterization of TdTrxh2 through its expression in yeast cells and Arabidopsis plants. Sequence analysis revealed that TdTrxh2 protein shared the conserved redox site with the other Trxh from other plant species. Under various abiotic stresses, TdTrxh2 was up-regulated in leaves and roots of durum wheat. Interestingly, we demonstrated that TdTrxh2 exhibit protective effect on LDH activity against various treatments. Besides, the expression of TdTrxh2 in yeast cells conferred their tolerance to multiple stresses. Moreover, transgenic Arabidopsis expressing TdTrxh2 showed tolerance phenotype to several abiotic stresses. This tolerance was illustrated by high rate of proline accumulation, root proliferation, low accumulation of reactive oxygen species like H2O2 and O2·-, and high antioxidant CAT and POD enzymes activities. All these findings suggested that TdTrxh2 promotes abiotic stress tolerance through the redox homoeostasis regulation and its protective role.

The long noncoding RNAs lnc10 and lnc11 regulating flavonoid biosynthesis in Ginkgo biloba.

Although long non-coding RNAs have been recognized to play important roles in plant, their possible functions and potential mechanism in Ginkgo biloba flavonoid biosynthesis are poorly understood. Flavonoids are important secondary metabolites and healthy components of Ginkgo biloba. They have been widely used in food, medicine, and natural health products. Most previous studies have focused on the molecular mechanisms of structural genes and transcription factors that regulate flavonoid biosynthesis. Few reports have examined the biological functions of flavonoid biosynthesis by long non-coding RNAs in G. biloba. Long noncoding RNAs associated with flavonoid biosynthesis in G. biloba have been identified through RNA sequencing, but the function of lncRNAs has not been reported. In this study, the expression levels of lnc10 and lnc11 were identified. Quantitative real-time polymerase chain reaction analysis revealed that lnc10 and lnc11 were expressed in all detected organs, and they showed significantly higher levels in immature and mature leaves than in other organs. In addition, to fully identify the function of lnc10 and lnc11 in flavonoid biosynthesis in G. biloba, lnc10 and lnc11 were cloned from G. biloba, and were transformed into Arabidopsis and overexpressed. Compared with the wild type, the flavonoid content was increased in transgenic plants. Moreover, the RNA-sequencing analysis of wild-type, lnc10-overexpression, and lnc11-overexpression plants screened out 2019 and 2552 differentially expressed genes, and the transcript levels of structural genes and transcription factors associated with flavonoid biosynthesis were higher in transgenic Arabidopsis than in the wild type, indicating that lnc10 and lnc11 activated flavonoid biosynthesis in the transgenic lines. Overall, these results suggest that lnc10 and lnc11 positively regulate flavonoid biosynthesis in G. biloba.

Overexpression of KvCHX Enhances Salt Tolerance in Arabidopsis thaliana Seedlings.

The CHX (cation/H+ exchanger) family plays an important role in the transmembrane transport of cation/H+ in plants. The aim of this study was to identify and functionally analyze the KvCHX gene in the halophyte Kosteletzkya virginica to investigate its role in regulating the K+/Na+ ratio under salinity tolerance. Based on a partial gene sequence of EST from K. virginica, the full-length DNA sequence of the KvCHX gene was obtained using genome walking technology. Structural analysis and phylogenetic relationship analysis showed that the KvCHX gene was closely related to the AtCHX17 gene. The KvCHX overexpression vector was successfully constructed and transformed into Arabidopsis via floral dipping. Arabidopsis seedlings overexpressing KvCHX showed an enhanced tolerance to salt stress compared with wild-type plants. Transgenic Arabidopsis seedlings grew better under K+ deficiency than WT. The results showed that KvCHX could promote the uptake of K+, increase the ratio of K+/Na+, and promote the growth of plants under K+ deficiency and treatment with NaCl solution. KvCHX is involved in K+ transport and improves plant salt tolerance by coordinating K+ acquisition and homeostasis.

Functional characterization of a cold related flavanone 3-hydroxylase from Tetrastigma hemsleyanum: an in vitro, in silico and in vivo study.

Tetrastigma hemsleyanum Diels et Gilg, a traditional Chinese medicine, frequently suffers from cold damage in the winter, leading to lower yields. There is a pressing need to improve cold resistance; however, the mechanisms underlying T. hemsleyanum responses to cold stress are still not clearly understood. Here, we explored the function of the flavanone 3-hydroxylase gene (ThF3H) in T. hemsleyanum under cold treatment. The open reading frame of ThF3H is 1092 bp and encodes 363 amino acid residues. In vitro, the ThF3H enzyme was expressed in E. coli and successfully catalyzed naringenin and eriodictyol into dihydrokaempferol and dihydroquercetin, respectively. ThF3H exhibited a higher affinity for naringenin than for eriodictyol, which was in accordance with an in silico molecular docking analysis. The optimal pH and temperature for ThF3H activity were 7.0 and 30 °C, respectively. In vivo, overexpression of the ThF3H gene enhanced the cold tolerance of transgenic Arabidopsis lines, which was likely due to the increase in flavonoids. Collectively, the function of a cold-related ThF3H in the flavonoid biosynthesis pathway may be helpful for improving the cold tolerance of T. hemsleyanum through molecular breeding techniques.

Cloning and functional analysis of the BrCUC2 gene in Brassica rapa L.

The CUP-SHAPED COTYLEDON2 (CUC2) gene plays an important role in the formation of apical meristem and organ edges in plants. The apical meristematic tissue of Brassica rapa (B. rapa) is associated with cold resistance, however, the role of the CUC2 gene in cold resistance of B.rapa is unclear. In this study, we used bioinformatics software to analyze the structure of BrCUC2 gene, real-time fluorescence quantitative PCR to detect the expression level of BrCUC2, constructed transgenic Arabidopsis thaliana by the flower dipping method and subcellular localization for functional validation. The results showed that, we isolated a 1104 bp open reading frame of BrCUC2 from the winter B. rapa cultivar 'Longyou 7'. The BrCUC2 contains a highly conserved domain belonging to the NAM superfamily. Its homologus CUC genes contain similar conserved motifs and are closely related to Brassica oleracea (B.oleracea), and the N-terminal of amino acid sequence contains NAC domain. BrCUC2 protein was localized in the nucleus and self-activation tests showed that pGBKT7-BrCUC2 had self-activation. Tissue-specific expression analysis and promoter ß-Glucuronidase (GUS) activity showed that BrCUC2 had high expression levels in B. rapa growth points and A. thaliana leaf edges, stems and growth points. After low-temperature stress, BrCUC2 showed greater expression in 'Longyou 7,' which presents strong cold resistance and concave growth points, than in 'Longyou 99,' which presents weak cold resistance and protruding growth points. BrCUC2 promoter contains multiple elements related to stress responses. BrCUC2 overexpression revealed that the phenotype did not differ from that of the wild type during the seedling stage but showed weak growth and a dwarf phenotype during the flowering and mature stages. After low-temperature treatment, the physiological indexes and survival rate of BrCUC2-overexpression lines of Arabidopsis thaliana (A. thaliana) were better than those of the wild type within 12 h, although differences were not observed after 24 h. These results showed that BrCUC2 improved the low-temperature tolerance of transgenic A. thaliana within a short time. It can provide a foundation for the study of cold resistance in winter B. rapa.

Functional Characterisation of the Transcription Factor GsWRKY23 Gene from Glycine soja in Overexpressed Soybean Composite Plants and Arabidopsis under Salt Stress.

WRKY proteins are a superfamily of transcription factors (TFs) that play multiple roles in plants' growth, development, and environmental stress response. In this study, a novel WRKY gene called GsWRKY23 that is specifically upregulated in salt-tolerant Glycine soja accession BB52 seedlings was identified by transcriptomic analysis under salt stress. How the physiological functions and mechanisms of the GsWRKY23 gene affect salt tolerance was investigated using transformations of soybean hairy roots and Arabidopsis, including wild-type (WT) and atwrky23-mutant plants. The results showed that GsWRKY23 in the roots, stems, and leaves of BB52, along with its promoter in the cotyledons and root tips of GsWRKY23pro::GUS Arabidopsis seedlings, displayed enhanced induction under salt stress. GsWRKY23 localises to the nucleus and shows transcriptional activation ability in yeast cells. Compared to GsWRKY23-RNAi wild soybean hairy-root composite plants under salt stress, obvious improvements, such as superior growth appearance, plant height and fresh weight (FW), and leaf chlorophyll and relative water content (RWC), were displayed by GsWRKY23-overexpressing (OE) composite plants. Moreover, their relative electrolytic leakage (REL) values and malondialdehyde (MDA) contents in the roots and leaves declined significantly. Most of the contents of Na+ and Cl- in the roots, stems, and leaves of GsWRKY23-OE plants decreased significantly, while the content of K+ in the roots increased, and the content of NO3- displayed no obvious change. Ultimately, the Na+/K+ ratios of roots, stems, and leaves, along with the Cl-/NO3- ratios of roots and stems, decreased significantly. In the transgenic WT-GsWRKY23 and atwrky23-GsWRKY23 Arabidopsis seedlings, the salt-induced reduction in seed germination rate and seedling growth was markedly ameliorated; plant FW, leaf chlorophyll content, and RWC increased, and the REL value and MDA content in shoots decreased significantly. In addition, the accumulation of Na+ and Cl- decreased, and the K+ and NO3- levels increased markedly to maintain lower Na+/K+ and Cl-/NO3- ratios in the roots and shoots. Taken together, these results highlight the role of GsWRKY23 in regulating ionic homeostasis in NaCl-stressed overexpressed soybean composite plants and Arabidopsis seedlings to maintain lower Na+/K+ and Cl-/NO3- ratios in the roots and shoots, thus conferring improved salt tolerance.

Cloning and functional characterization of FtWRKY29, a low phosphorus stress-inducible transcription factor in Tartary buckwheat.

The regulation of the expression of genes related to abiotic stress in plants is significantly influenced by the binding of the transcription factor (TF) WRKY to the W-box elements in their promoters. The findings of this study have confirmed that the ability of Tartary buckwheat (Fagopyrum tataricum Gaertn.) to tolerate phosphorus (P) deficiency is regulated by FtWRKY29, which is classified as a member of group II of the WRKY family. The roots predominantly exhibited an enhanced expression of FtWRKY29, which was significantly upregulated in response to low-P-induced stress. The W-box motif was bound to by FtWRKY29 which enhanced the transcription of genes and was localized to the nucleus. The overexpression of FtWRKY29 in Arabidopsis thaliana produced transgenic lines that exhibited phenotypes typical of diminished sensitivity to low-P-induced stress by promoting root growth, increasing P-uptake, and regulating the accumulation of anthocyanin. The low-P-responsive genes, PHT1;1, PHT1;4, and PHO1 were significantly up-regulated in these lines. In addition, the overexpression of FtWRKY29 restored the P-absorption ability of the wrky75 mutant to a certain extent. Moreover, the binding of FtWRKY29 to the promoter of PHT1;1 activated its expression in tobacco. It was also observed that FtWRKY29 interacts with AtMPK3, AtMPK6, FtMPK3, and FtMPK7. This study provides preliminary evidence that FtWRKY29 improved the tolerance of transgenic A. thaliana plants to low-P-induced stress and deepened the understanding of the regulatory mechanism behind the same in Tartary buckwheat.

A Transcription Factor SlNAC4 Gene of Suaeda liaotungensis Enhances Salt and Drought Tolerance through Regulating ABA Synthesis.

The NAC (NAM, ATAF1/2 and CUC2) transcription factors are ubiquitously distributed in plants and play critical roles in the construction of plant organs and abiotic stress response. In this study, we described the cloning of a Suaeda liaotungensis K. NAC transcription factor gene SlNAC4, which contained 1450 bp, coding a 331 amino acid. We found that SlNAC4 was highly expressed in stems of S. liaotungensis, and the expression of SlNAC4 was considerably up-regulated after salt, drought, and ABA treatments. Transcription analysis and subcellular localization demonstrated that the SlNAC4 protein was located both in the nucleus and cytoplasm, and contained a C-terminal transcriptional activator. The SlNAC4 overexpression Arabidopsis lines significantly enhanced the tolerance to salt and drought treatment and displayed obviously increased activity of antioxidant enzymes under salt and drought stress. Additionally, transgenic plants overexpressing SlNAC4 had a significantly higher level of physiological indices. Interestingly, SlNAC4 promoted the expression of ABA metabolism-related genes including AtABA1, AtABA3, AtNCED3, AtAAO3, but inhibited the expression of AtCYP707A3 in overexpression lines. Using a yeast one-hybrid (Y1H) assay, we identified that the SlNAC4 transcription factor could bind to the promoters of those ABA metabolism-related genes. These results indicate that overexpression of SlNAC4 in plants enhances the tolerance to salt and drought stress by regulating ABA metabolism.

Biological function research of Fusarium oxysporum f. sp. cubense inducible banana long noncoding RNA Malnc2310 in Arabidopsis.

Long noncoding RNAs (lncRNAs) participate in plant biological processes under biotic and abiotic stresses. However, little is known about the function and regulation mechanism of lncRNAs related to the pathogen at a molecular level. A banana lncRNA, Malnc2310, is a Fusarium oxysporum f. sp. cubense inducible lncRNA in roots. In this study, we demonstrate the nuclear localization of Malnc2310 by fluorescence in situ hybridization and it can bind to several proteins that are related to flavonoid pathway, pathogen response and programmed cell death. Overexpression of Malnc2310 increases susceptibility to Fusarium crude extract (Fu), salinity, and cold in transgenic Arabidopsis. In addition, Malnc2310 transgenic Arabidopsis accumulated more anthocyanins under Fusarium crude extract and cold treatments that are related to upregulation of these genes involved in anthocyanin biosynthesis. Based on our findings, we propose that Malnc2310 may participate in flavonoid metabolism in plants under stress. Furthermore, phenylalanine ammonia lyase (PAL) protein expression was enhanced in Malnc2310 overexpressed transgenic Arabidopsis, and Malnc2310 may participate in PAL regulation by binding to it. This study provides new insights into the role of Malnc2310 in mediating plant stress adaptation.

Overexpression of peanut (Arachis hypogaea L.) AhGRFi gene enhanced root growth inhibition under exogenous NAA treatment in Arabidopsis thaliana.

The 14-3-3 protein is a kind of evolutionary ubiquitous protein family highly conserved in eukaryotes. Initially, 14-3-3 proteins were reported in mammalian nervous tissues, but in the last decade, their role in various metabolic pathways in plants established the importance of 14-3-3 proteins. In the present study, a total of 22 14-3-3 genes, also called general regulatory factors (GRF), were identified in the peanut (Arachis hypogaea) genome, out of which 12 belonged to the ε group, whereas 10 of them belonged to the non- ε-group. Tissue-specific expression of identified 14-3-3 genes were studied using transcriptome analysis. The peanut AhGRFi gene was cloned and transformed into Arabidopsis thaliana. The investigation of subcellular localization indicated that AhGRFi is localized in the cytoplasm. Overexpression of the AhGRFi gene in transgenic Arabidopsis showed that under exogenous 1-naphthaleneacetic acid (NAA) treatment, root growth inhibition in transgenic plants was enhanced. Further analysis indicated that the expression of auxin-responsive genes IAA3, IAA7, IAA17, and SAUR-AC1 was upregulated and GH3.2 and GH3.3 were downregulated in transgenic plants, but the expression of GH3.2, GH3.3, and SAUR-AC1 showed opposite trends of change under NAA treatment. These results suggest that AhGRFi may be involved in auxin signaling during seedling root development. An in-depth study of the molecular mechanism of this process remains to be further explored.

Unraveling the functional characterization of a jasmonate-induced flavonoid biosynthetic CYP45082G24 gene in Carthamus tinctorius.

The cytochrome P450 superfamily of monooxygenases plays a major role in the evolution and diversification of plant natural products. The function of cytochrome P450s in physiological adaptability, secondary metabolism, and xenobiotic detoxification has been studied extensively in numerous plant species. However, their underlying regulatory mechanism in safflower still remained unclear. In this study, we aimed to elucidate the functional role of a putative CtCYP82G24-encoding gene in safflower, which suggests crucial insights into the regulation of methyl jasmonate-induced flavonoid accumulation in transgenic plants. The results showed that methyl jasmonate (MeJA) was associated with a progressive upregulation of CtCYP82G24 expression in safflower among other treatment conditions including light, dark, and polyethylene glycol (PEG). In addition, transgenic plants overexpressing CtCYP82G24 demonstrated increased expression level of other key flavonoid biosynthetic genes, such as AtDFR, AtANS, and AtFLS, and higher content of flavonoid and anthocyanin accumulation when compared with wild-type and mutant plants. Under exogenous MeJA treatment, the CtCYP82G24 transgenic overexpressed lines showed a significant spike in flavonoid and anthocyanin content compared with wild-type and mutant plants. Moreover, the virus-induced gene silencing (VIGS) assay of CtCYP82G24 in safflower leaves exhibited decreased flavonoid and anthocyanin accumulation and reduced expression of key flavonoid biosynthetic genes, suggesting a possible coordination between transcriptional regulation of CtCYP82G24 and flavonoid accumulation. Together, our findings confirmed the likely role of CtCYP82G24 during MeJA-induced flavonoid accumulation in safflower.

Heterologous expression of MirMAN enhances root development and salt tolerance in Arabidopsis.

Introduction: ß-Mannanase is a plant cell wall remodeling enzyme involved in the breakdown of hemicellulose and plays an important role in growth by hydrolyzing the mannan-like polysaccharide, but its function in adaptation to salt stress has been less studied. Methods: Based on cloned the mannanase (MAN) gene from Mirabilis jalapa L., the study was carried out by heterologously expressing the gene in Arabidopsis thaliana, and then observing the plant phenotypes and measuring relevant physiological and biochemical indicators under 150 mM salt treatment. Results and discussion: The results indicate that MirMAN is a protein with a glycohydrolase-specific structural domain located in the cell wall. We first found that MirMAN reduced the susceptibility of transgenic Arabidopsis thaliana to high salt stress and increased the survival rate of plants by 38%. This was corroborated by the following significant changes, including the reduction in reactive oxygen species (ROS) levels, increase in antioxidant enzyme activity, accumulation of soluble sugars and increase of the expression level of RD29 in transgenic plants. We also found thatthe heterologous expression of MirMAN promoted root growth mainly by elongating the primary roots and increasing the density of lateral roots. Meanwhile, the expression of ARF7, ARF19, LBD16 and LBD29 was up-regulated in the transgenic plants, and the concentration of IAA in the roots was increased. Those results indicate that MirMAN is involved in the initiation of lateral root primordia in transgenic plants through the IAA-ARF signalling pathway. In conclusion, MirMAN improves plant salt tolerance not only by regulating ROS homeostasis, but also by promoting the development of lateral roots. Reflecting the potential of the MirMAN to promote root plastic development in adaptation to salt stress adversity.

GmABR1 encoding an ERF transcription factor enhances the tolerance to aluminum stress in Arabidopsis thaliana.

The ethylene response factor (ERF) transcription factors, which is one of the largest transcription factor families in plants, are involved in biological and abiotic stress response and play an important role in plant growth and development. In this study, the GmABR1 gene from the soybean inbred line Zhonghuang24 (ZH24)×Huaxia 3 (HX3) was investigated its aluminum (Al) tolerance. GmABR1 protein has a conserved domain AP2, which is located in the nucleus and has transcriptional activation ability. The results of real-time quantitative PCR (qRT-PCR) showed that the GmABR1 gene presented a constitutive expression pattern rich in the root tip, stem and leaf tissues of HX3. After Al stress, the GmABR1 transcript was significantly increased in the roots. The transcripts of GmABR1 in the roots of HX3 treated with 50 µM AlCl3 was 51 times than that of the control. The GmABR1 was spatiotemporally specific with the highest expression levels when Al concentration was 50 µM, which was about 36 times than that of the control. The results of hematoxylin staining showed that the root tips of GmABR1-overexpression lines were stained the lightest, followed by the control, and the root tips of GmABR1 RNAi lines were stained the darkest. The concentrations of Al3+ in root tips were 207.40 µg/g, 147.74 µg/g and 330.65 µg/g in wild type (WT), overexpressed lines and RNAi lines, respectively. When AlCl3 (pH4.5) concentration was 100 µM, all the roots of Arabidopsis were significantly inhibited. The taproot elongation of WT, GmABR1 transgenic lines was 69.6%, 85.6%, respectively. When treated with Al, the content of malondialdehyde (MDA) in leaves of WT increased to 3.03 µg/g, while that of transgenic Arabidopsis increased from 1.66-2.21 µg/g, which was lower than that of WT. Under the Al stress, the Al stress responsive genes such as AtALMT1 and AtMATE, and the genes related to ABA pathway such as AtABI1, AtRD22 and AtRD29A were up-regulated. The results indicated that GmABR1 may jointly regulate plant resistance to Al stress through genes related to Al stress response and ABA response pathways.