Comprehensive identification of maize ZmE2F transcription factors and the positive role of ZmE2F6 in response to drought stress.

BACKGROUND: The early 2 factor (E2F) family is characterized as a kind of transcription factor that plays an important role in cell division, DNA damage repair, and cell size regulation. However, its stress response has not been well revealed. RESULTS: In this study, ZmE2F members were comprehensively identified in the maize genome, and 21 ZmE2F genes were identified, including eight E2F subclade members, seven DEL subfamily genes, and six DP genes. All ZmE2F proteins possessed the DNA-binding domain (DBD) characterized by conserved motif 1 with the RRIYD sequence. The ZmE2F genes were unevenly distributed on eight maize chromosomes, showed diversity in gene structure, expanded by gene duplication, and contained abundant stress-responsive elements in their promoter regions. Subsequently, the ZmE2F6 gene was cloned and functionally verified in drought response. The results showed that the ZmE2F6 protein interacted with ZmPP2C26, localized in the nucleus, and responded to drought treatment. The overexpression of ZmE2F6 enhanced drought tolerance in transgenic Arabidopsis with longer root length, higher survival rate, and biomass by upregulating stress-related gene transcription. CONCLUSIONS: This study provides novel insights into a greater understanding and functional study of the E2F family in the stress response.

Overexpression of NtEXPA7 promotes seedling growth and resistance to root-knot nematode in tobacco.

Root-knot nematode (RKN) is one of the most damaging plant pathogen in the world. They exhibit a wide host range and cause serious crop losses. The cell wall, encasing every plant cell, plays a crucial role in defending of RKN invasion. Expansins are a group of cell wall proteins inducing cell wall loosening and extensibility. They are widely involved in the regulation of plant growth and the response to biotic and abiotic stresses. In this study, we have characterized the biological function of tobacco (Nicotiana tabacum) NtEXPA7, the homologue of Solyc08g080060.2 (SlEXPA18), of which the transcription level was significantly reduced in susceptible tomato upon RKN infection. The expression of NtEXPA7 was up-regulated after inoculation of RKNs. The NtEXPA7 protein resided in the cell wall. Overexpression of NtEXPA7 promoted the seedling growth of transgenic tobacco. Meanwhile the increased expression of NtEXPA7 was beneficial to enhance the resistance against RKNs. This study expands the understanding of biological role of expansin in coordinate plant growth and disease resistance.

Maize ZmLAZ1-3 gene negatively regulates drought tolerance in transgenic Arabidopsis.

BACKGROUND: Molecular mechanisms in response to drought stress are important for the genetic improvement of maize. In our previous study, nine ZmLAZ1 members were identified in the maize genome, but the function of ZmLAZ1 was largely unknown. RESULTS: The ZmLAZ1-3 gene was cloned from B73, and its drought-tolerant function was elucidated by expression analysis in transgenic Arabidopsis. The expression of ZmLAZ1-3 was upregulated by drought stress in different maize inbred lines. The driving activity of the ZmLAZ1-3 promoter was induced by drought stress and related to the abiotic stress-responsive elements such as MYB, MBS, and MYC. The results of subcellular localization indicated that the ZmLAZ1-3 protein localized on the plasma membrane and chloroplast. The ectopic expression of the ZmLAZ1-3 gene in Arabidopsis significantly reduced germination ratio and root length, decreased biomass, and relative water content, but increased relative electrical conductivity and malondialdehyde content under drought stress. Moreover, transcriptomics analysis showed that the differentially expressed genes between the transgenic lines and wild-type were mainly associated with response to abiotic stress and biotic stimulus, and related to pathways of hormone signal transduction, phenylpropanoid biosynthesis, mitogen-activated protein kinase signaling, and plant-pathogen interaction. CONCLUSION: The study suggests that the ZmLAZ1-3 gene is a negative regulator in regulating drought tolerance and can be used to improve maize drought tolerance via its silencing or knockout.

Comprehensive Identification of the Pum Gene Family and Its Involvement in Kernel Development in Maize.

The Pumilio (Pum) RNA-binding protein family regulates post-transcription and plays crucial roles in stress response and growth. However, little is known about Pum in plants. In this study, a total of 19 ZmPum genes were identified and classified into two groups in maize. Although each ZmPum contains the conserved Pum domain, the ZmPum members show diversity in the gene and protein architectures, physicochemical properties, chromosomal location, collinearity, cis-elements, and expression patterns. The typical ZmPum proteins have eight α-helices repeats, except for ZmPum2, 3, 5, 7, and 14, which have fewer α-helices. Moreover, we examined the expression profiles of ZmPum genes and found their involvement in kernel development. Except for ZmPum2, ZmPum genes are expressed in maize embryos, endosperms, or whole seeds. Notably, ZmPum4, 7, and 13 exhibited dramatically high expression levels during seed development. The study not only contributes valuable information for further validating the functions of ZmPum genes but also provides insights for improvement and enhancing maize yield.

Molecular and Functional Analysis of Trehalose-6-Phosphate Synthase Genes Enhancing Salt Tolerance in Anoectochilus roxburghii (Wall.) Lindl.

Trehalose is a reducing disaccharide, acting as a protectant against various environmental stresses in numerous organisms. In plants, trehalose-6-phosphate synthase (TPS) plays a crucial role in trehalose biosynthesis. Anoectochilus roxburghii (Wall.) Lindl. is a prominent species of the Anoectochilus genus, widely utilized as a health food. However, the functional analysis of TPS in this species has been limited. In this study, TPS genes were cloned from A. roxburghii. The ArTPS gene, with an open reading frame spanning 2850 bp, encodes 950 amino acids. Comparative and bioinformatics analysis revealed that the homology was presented between the ArTPS protein and TPSs from other plant species. The ORF sequence was utilized to construct a prokaryotic expression vector, Pet28a-ArTPS, which was then transformed into Escherichia coli. The resulting transformants displayed a significant increase in salt tolerance under the stress conditions of 300 mmol/L NaCl. Quantitative RT-PCR analysis demonstrated that the expression of ArTPS genes responded to NaCl stress. The accumulation of G6P was upregulated, whereas the content of T6P exhibited an opposite expression trend. The glycometabolism products, including trehalose, exhibited notable changes under NaCl stress, although their variations may differ in response to stimulation. The content of kinsenoside, a characteristic product of A. roxburghii, was significantly upregulated under NaCl stress. These results suggest that the ArTPS genes function in response to NaCl stimulation and play a key role in polysaccharide and glycoside metabolism in Anoectochilus. This study provides new insights into the engineering modification of the health food A. roxburghii to enhance the medicinal activity of its ingredients.

Transgenic Improvement for Biotic Resistance of Crops.

Biotic constraints, including pathogenic fungi, viruses and bacteria, herbivory insects, as well as parasitic nematodes, cause significant yield loss and quality deterioration of crops. The effect of conventional management of these biotic constraints is limited. The advances in transgenic technologies provide a direct and directional approach to improve crops for biotic resistance. More than a hundred transgenic events and hundreds of cultivars resistant to herbivory insects, pathogenic viruses, and fungi have been developed by the heterologous expression of exogenous genes and RNAi, authorized for cultivation and market, and resulted in a significant reduction in yield loss and quality deterioration. However, the exploration of transgenic improvement for resistance to bacteria and nematodes by overexpression of endogenous genes and RNAi remains at the testing stage. Recent advances in RNAi and CRISPR/Cas technologies open up possibilities to improve the resistance of crops to pathogenic bacteria and plant parasitic nematodes, as well as other biotic constraints.

Genome-Wide Characterization and Function Analysis of ZmERD15 Genes' Response to Saline Stress in Zea mays L.

Early responsive dehydration (ERD) genes can be rapidly induced by dehydration. ERD15 genes have been confirmed to regulate various stress responses in plants. However, the maize ERD15 members have not been characterized. In the present study, a total of five ZmERD15 genes were identified from the maize genome and named ZmERD15a, ZmERD15b, ZmERD15c, ZmERD15d, and ZmERD15e. Subsequently, their protein properties, gene structure and duplication, chromosomal location, cis-acting elements, subcellular localization, expression pattern, and over-expression in yeast were analyzed. The results showed that the ZmERD15 proteins were characterized by a similar size (113-159 aa) and contained a common domain structure, with PAM2 and adjacent PAE1 motifs followed by an acidic region. The ZmERD15 proteins exhibited a close phylogenetic relationship with OsERD15s from rice. Five ZmERD15 genes were distributed on maize chromosomes 2, 6, 7, and 9 and showed a different exon-intron organization and were expanded by duplication. Besides, the promoter region of the ZmERD15s contained abundant cis-acting elements that are known to be responsive to stress and hormones. Subcellular localization showed that ZmERD15b and ZmERD15c were localized in the nucleus. ZmERD15a and ZmERD15e were localized in the nucleus and cytoplasm. ZmERD15d was localized in the nucleus and cell membrane. The results of the quantitative real-time PCR (qRT-PCR) showed that the expression of the ZmERD15 genes was regulated by PEG, salinity, and ABA. The heterologous expression of ZmERD15a, ZmERD15b, ZmERD15c, and ZmERD15d significantly enhanced salt tolerance in yeast. In summary, a comprehensive analysis of ZmERD15s was conducted in the study. The results will provide insights into further dissecting the biological function and molecular mechanism of ZmERD15s regulating of the stress response in maize.

Comprehensive Identification and Functional Analysis of Stress-Associated Protein (SAP) Genes in Osmotic Stress in Maize.

Stress-associated proteins (SAPs) are a kind of zinc finger protein with an A20/AN1 domain and contribute to plants' adaption to various abiotic and biological stimuli. However, little is known about the SAP genes in maize (Zea mays L.). In the present study, the SAP genes were identified from the maize genome. Subsequently, the protein properties, gene structure and duplication, chromosomal location, and cis-acting elements were analyzed by bioinformatic methods. Finally, their expression profiles under osmotic stresses, including drought and salinity, as well as ABA, and overexpression in Saccharomyces cerevisiae W303a cells, were performed to uncover the potential function. The results showed that a total of 10 SAP genes were identified and named ZmSAP1 to ZmSAP10 in maize, which was unevenly distributed on six of the ten maize chromosomes. The ZmSAP1, ZmSAP4, ZmSAP5, ZmSAP6, ZmSAP7, ZmSAP8 and ZmSAP10 had an A20 domain at N terminus and AN1 domain at C terminus, respectively. Only ZmSAP2 possessed a single AN1 domain at the N terminus. ZmSAP3 and ZmSAP9 both contained two AN1 domains without an A20 domain. Most ZmSAP genes lost introns and had abundant stress- and hormone-responsive cis-elements in their promoter region. The results of quantitative real-time PCR showed that all ZmSAP genes were regulated by drought and saline stresses, as well as ABA induction. Moreover, heterologous expression of ZmSAP2 and ZmSAP7 significantly improved the saline tolerance of yeast cells. The study provides insights into further underlying the function of ZmSAPs in regulating stress response in maize.

Maize ZmBES1/BZR1-3 and -9 Transcription Factors Negatively Regulate Drought Tolerance in Transgenic Arabidopsis.

The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1(BZR1) transcription factors play crucial roles in plant growth, development, and stress response. However, little is known about the function of maize's BES1/BZR1s. In this study, the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes were cloned from maize's inbred line, B73, and they were functionally evaluated by analyzing their expression pattern, subcellular localization, transcriptional activation activity, as well as their heterologous expression in Arabidopsis, respectively. The results of the qRT-PCR showed that the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes were predominantly expressed in the root, and their expression was significantly down-regulated by drought stress. The ZmBES1/BZR1-3 and ZmBES1/BZR1-9 proteins localized in the nucleus but showed no transcriptional activation activity as a monomer. Subsequently, it was found that the heterologous expression of the ZmBES1/BZR1-3 and ZmBES1/BZR1-9 genes in Arabidopsis decreased drought tolerance, respectively. The transgenic lines showed a more serious wilting phenotype, shorter root length, lower fresh weight, and higher relative electrolyte leakage (REL) and malondialdehyde (MDA) content compared to the control under drought stress. The RNA-sequencing data showed that the 70.67% and 93.27% differentially expressed genes (DEGs) were significantly down-regulated in ZmBES1/BZR1-3 and ZmBES1/BZR1-9 transgenic Arabidopsis, respectively. The DEGs of ZmBES1/BZR1-3 gene's expressing lines were mainly associated with oxidative stress response and amino acid metabolic process and enriched in phenylpropanoid biosynthesis and protein processing in the endoplasmic reticulum. But the DEGs of the ZmBES1/BZR1-9 gene's expressing lines were predominantly annotated with water deprivation, extracellular stimuli, and jasmonic acid and enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. Moreover, ZmBES1/BZR1-9 increased stomatal aperture in transgenic Arabidopsis under drought stress. This study indicates that ZmBES1/BZR1-3 and ZmBES1/BZR1-9 negatively regulate drought tolerance via different pathways in transgenic Arabidopsis, and it provides insights into the underlying the function of BES1/BZR1s in crops.

Maize transcription factor ZmBES1/BZR1-5 positively regulates kernel size.

The BES1/BZR1 transcription factors regulate the expression of genes responsive to brassinosteroids and play pivotal roles in plant development, but their role in regulating kernel development in maize remains unclear. In this study, we found that ZmBES1/BZR1-5 positively regulates kernel size. Association analysis of candidate genes in 513 diverse maize inbred lines indicated that three SNPs related to ZmBES1/BZR1-5 were significantly associated with kernel width and whilst four SNPs were related to 100-kernel weight. Overexpression of ZmBES1/BZR1-5 in Arabidopsis and rice both significantly increased seed size and weight, and smaller kernels were produced in maize Mu transposon insertion and EMS mutants. The ZmBES1/BZR1-5 protein locates in the nucleus, contains bHLH and BAM domains, and shows no transcriptional activity as a monomer but forms a homodimer through the BAM domain. ChIP-sequencing analysis, and yeast one-hybrid and dual-luciferase assays demonstrated that the protein binds to the promoters of AP2/EREBP genes (Zm00001d010676 and Zm00001d032077) and inhibits their transcription. cDNA library screening showed that ZmBES1/BZR1-5 interacts with casein kinase II subunit ß4 (ZmCKIIß4) and ferredoxin 2 (ZmFdx2) in vitro and in vivo, respectively. Taken together, our study suggests that ZmBES1/BZR1-5 positively regulates kernel size, and provides new insights into understanding the mechanisms of kernel development in maize.

Maize ZmBES1/BZR1-5 Decreases ABA Sensitivity and Confers Tolerance to Osmotic Stress in Transgenic Arabidopsis.

The BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors, key components in the brassinosteroid signaling pathway, play pivotal roles in plant growth and development. However, the function of BES1/BZR1 in crops during stress response remains poorly understood. In the present study, we characterized ZmBES1/BZR1-5 from maize, which was localized to the nucleus and was responsive to abscisic acid (ABA), salt and drought stresses. Heterologous expression of ZmBES1/BZR1-5 in transgenic Arabidopsis resulted in decreased ABA sensitivity, facilitated shoot growth and root development, and enhanced salt and drought tolerance with lower malondialdehyde (MDA) content and relative electrolyte leakage (REL) under osmotic stress. The RNA sequencing (RNA-seq) analysis revealed that 84 common differentially expressed genes (DEGs) were regulated by ZmBES1/BZR1-5 in transgenic Arabidopsis. Subsequently, gene ontology and KEGG pathway enrichment analyses showed that the DEGs were enriched in response to stress, secondary metabolism and metabolic pathways. Furthermore, 30 DEGs were assigned to stress response and possessed 2-15 E-box elements in their promoters, which could be potentially recognized and bound by ZmBES1/BZR1-5. Taken together, our results reveal that the ZmBES1/BZR1-5 transcription factor positively regulates salt and drought tolerance by binding to E-box to induce the expression of downstream stress-related genes. Therefore, our study contributes to the better understanding of BES1/BZR1 function in the stress response of plants.

Isolation and characterization of maize ZmPP2C26 gene promoter in drought-response.

The clade A members of serine/threonine protein phosphatase 2Cs (PP2Cs) play crucial roles in plant growth, development, and stress response via the ABA signaling pathway. But little is known about other PP2C clades in plants. Our previous study showed that maize the ZmPP2C26, a clade B member of ZmPP2Cs, negatively regulated drought tolerance in transgenic Arabidopsis. However, the upstream regulatory mechanism of ZmPP2C26 remains unclear. In the present study, the expression of ZmPP2C26 gene in maize was analyzed by quantitative real time PCR (qRT-PCR). The results showed that the expression of ZmPP2C26 in shoot and root was both significantly inhibited by drought stress. Subsequently, a 2175 bp promoter of ZmPP2C26 was isolated from maize genome (P 2175). To validate whether the promoter possess some key cis-element and negatively drive ZmPP2C26 expression in drought stress, three 5´-deletion fragments of 1505, 1084 and 215 bp was amplified from P 2175 and were fused to ß-glucuronidase (GUS) and luciferase gene (LUC) to produce promoter::GUS and promoter::LUC constructs, and transformed into tobacco, respectively. Transient expression assays indicated that all promoters could drive GUS and LUC expression. The GUS and LUC activity were both significantly inhibited by PEG-6000 treatment. Notably, the - 1084 to - 215 bp promoter possess one MBS element and inhibits the expression of GUS and LUC under drought stress. Meanwhile, we found that the 215 bp length is enough to drive ZmPP2C26 expression. These findings will provide insights into understanding the transcription-regulatory mechanism of ZmPP2C26 negatively regulating drought tolerance.

Isolation and identification of a vegetative organ-specific promoter from maize.

To avoid the unregulated overexpression of the exogenous genes, specific or inducible expression is necessary for some exogenous genes in transgenic plants. But little is known about organ- or tissue-specific promoters in maize. In the present study, the expression of a maize pentatricopeptide repeat (PPR) protein encoding gene, GRMZM2G129783, was analyzed by RNA-sequencing data and confirmed by quantitative real time PCR. The results showed that the PPR GRMZM2G129783 gene specifically expressed in vegetative organs. Consequently, a 1830 bp sequence upstream of the start codon of the promoter for GRMZM2G129783 gene was isolated from maize genome (P 1830 ). To validate whether the promoter possesses the vegetative organ-specificity, the full-length and three 5'-end deletion fragments of P 1830 of different length (1387, 437, and 146 bp) were fused with glucuronidase (GUS) gene to generate promoter::GUS constructs and transformed into tobacco. The transient expression and fluorometric GUS assay in transgenic tobacco showed that all promoter could drive the expression of the GUS gene, the - 437 to - 146 bp region possessed some crucial elements for root-specific expression, and the shortest and optimal sequence to maintain transcription activity was 146 and 437 bp in length, respectively. These results indicate that the promoter of the PPR GRMZM2G129783 gene is a vegetative organ-specific promoter and will be useful in transgenic modification of commercial crops for moderate specific expression after further evaluation in monocotyledons.

Interaction network of core ABA signaling components in maize.

KEY MESSAGE: We defined a comprehensive core ABA signaling network in monocot maize, including the gene expression, subcellular localization and interaction network of ZmPYLs, ZmPP2Cs, ZmSnRK2s and the putative substrates. The phytohormone abscisic acid (ABA) plays an important role in plant developmental processes and abiotic stress responses. In Arabidopsis, ABA is sensed by the PYL ABA receptors, which leads to binding of the PP2C protein phosphatase and activation of the SnRK2 protein kinases. These components functioning diversely and redundantly in ABA signaling are little known in maize. Using Arabidopsis pyl112458 and snrk2.2/3/6 mutants, we identified several ABA-responsive ZmPYLs and ZmSnRK2s, and also ZmPP2Cs. We showed the gene expression, subcellular localization and interaction network of ZmPYLs, ZmPP2Cs, and ZmSnRK2s, and the isolation of putative ZmSnRK2 substrates by mass spectrometry in monocot maize. We found that the ABA dependency of PYL-PP2C interactions is contingent on the identity of the PP2Cs. Among 238 candidate substrates for ABA-activated protein kinases, 69 are putative ZmSnRK2 substrates. Besides homologs of previously reported putative AtSnRK2 substrates, 23 phosphoproteins have not been discovered in the dicot Arabidopsis. Thus, we have defined a comprehensive core ABA signaling network in monocot maize and shed new light on ABA signaling.

The Small Auxin-Up RNA 50 (SAUR50) Gene from Ammopiptanthus nanus Negatively Regulates Drought Tolerance.

Drought stress is a primary abiotic stress that causes significant losses to forestry and agricultural production. Therefore, exploring drought-responsive genes and their regulatory mechanism is crucial for plant molecular breeding for forestry and agriculture production safety. Small auxin-up RNA (SAUR) proteins are essential in plant growth and development but show functional diversity in stress response. In this study, the transcriptome sequencing data of Ammopiptanthus nanus seedlings revealed that the expression of AnSAUR50 was continuously downregulated under drought stress. Hence, the AnSAUR50 gene was cloned and functionally analyzed in drought response. The results showed that the coding sequence of AnSAUR50 was 315 bp in length and encoded 104 amino acids. The AnSAUR50 protein showed high conservation, possessed a SAUR-specific domain, and localized in the nucleus and cell membrane. The heterologous expression of the AnSAUR50 gene enhanced the drought sensitivity of the transgenic Arabidopsis with a lower survival rate, biomass, and higher malondialdehyde content and relative electrolyte leakage. Moreover, transgenic plants showed shorter root lengths and bigger stomatal apertures, resulting in facilitating water loss under drought stress. The study indicates that AnSAUR50 negatively regulates drought tolerance by inhibiting root growth and stomatal closure, which provides insights into the underlying function and regulatory mechanism of SAURs in plant stress response.

Identification of novel natural anti-browning agents based on phenotypic and metabolites differences in potato cultivars.

This study aimed to elucidate the changes of browning-related metabolite in fresh-cut potato and to identify anti-browning agents. Metabolomics and weighted correlation network analysis (WGCNA) were used to identify metabolites and correlate them with potato browning traits. A total of 79 browning trait-positive-related metabolites and 19 browning trait-negative-related metabolites were obtained from four key modules via WGCNA. The accumulation of metabolites with rich reducing groups and acidic groups were found to enhance anti-browning activity in potatoes. Among these metabolites, only L-pyroglutamic acid (L-PA) and ascorbic acid had variable importance for the projection (VIP) values greater than 1.5. In addition, it was found that L-PA inhibited polyphenol oxidase (PPO) activity by lowering pH and interacting with amino acid residues of PPO. L-PA also inhibited the growth of microorganisms in fresh-cut potato. Our results show that L-AP is an effective novel anti-browning agent with antibacterial activity.

The Maize ZmBES1/BZR1-9 Transcription Factor Accelerates Flowering in Transgenic Arabidopsis and Rice.

In model plants, the BRI1-EMS suppressor 1 (BES1)/brassinazole-resistant 1 (BZR1) transcription factors play vital roles in regulating growth, development, and stimuli response. However, the roles of maize ZmBES1/BZR1 members are largely unknown. In this research, the ZmBES1/BZR1-9 gene was ectopically expressed in Arabidopsis and rice for the phenotyping of flowering. We found that the complementation and overexpression of ZmBES1/BZR1-9 in bes1-D mutant and wild type Arabidopsis both resulted in early flowering that was about 10 days shorter than in the untransformed control under long-day conditions. In addition, there was no difference in the rosette leaf number between all transgenic lines and the control. Subsequently, the ZmBES1/BZR1-9 gene was overexpressed in rice. It was found that overexpression lines of rice exhibited early flowering with heading dates that were 8 days shorter compared with untransformed plants. Moreover, the results of RNA-seq and qRT-PCR showed that five flowering-regulated genes, namely At2-MMP, AtPCC1, AtMYB56, AtPELPK1, and AtPRP10, were significantly up-regulated in all complementary and overexpressing lines of Arabidopsis. Meanwhile, the results of RNA-seq showed that 69 and 33 differentially expressed genes (DEGs) were up- and down-regulated in transgenic rice, respectively. Four flowering-related genes, namely OsGA20OX1, OsCCR19, OsBTBN19, and OsRNS4 were significantly up-regulated in transgenic lines. To sum up, our findings demonstrate that ZmBES1/BZR1-9 is involved in controlling flowering and provide insights into further underlying roles of BES1/BZR1s in regulating growth and development in crops.

Maize ZmBES1/BZR1-1 transcription factor negatively regulates drought tolerance.

Drought stress is a common abiotic factor and restricts plant growth and development. Exploring maize stress-related genes and their regulatory mechanisms is crucial for ensuring agricultural productivity and food security. The BRI1-EMS1 suppressor (BES1)/brassinazole-resistant 1 (BZR1) transcription factors (TFs) play important roles in plant growth, development, and stress response. However, maize ZmBES1/BZR1s are rarely reported. In the present study, the ZmBES1/BZR1-1 gene was cloned from maize B73 and functionally characterized in transgenic Arabidopsis and rice in drought stress response. The ZmBES1/BZR1-1 protein possessed a conserved bHLH domain characterized by BES1/BZR1 TFs, localized in the nucleus, and showed transcription activation activity. The expression of ZmBES1/BZR1-1 exhibited no tissue specificity but drought-inhibitory expression in maize. Under drought stress, overexpression of ZmBES1/BZR1-1 resulted in the enhancement of drought sensitivity of transgenic Arabidopsis and rice with a lower survival rate, reactive oxygen species (ROS) level and relative water content (RWC) and a higher stomatal aperture and relative electrolyte leakage (REL). The RNA-seq results showed that 56 differentially expressed genes (DEGs) were regulated by ZmBES1/BZR1-1 by binding to E-box elements in their promoters. The GO analysis showed that the DEGs were significantly annotated with response to oxidative stress and oxygen level. The study suggests that the ZmBES1/BZR1-1 gene negatively regulates drought stress, which provides insights into further underlying molecular mechanisms in the drought stress response mediated by BZR1/BES1s.

Three strategies of transgenic manipulation for crop improvement.

Heterologous expression of exogenous genes, overexpression of endogenous genes, and suppressed expression of undesirable genes are the three strategies of transgenic manipulation for crop improvement. Up to 2020, most (227) of the singular transgenic events (265) of crops approved for commercial release worldwide have been developed by the first strategy. Thirty-eight of them have been transformed by synthetic sequences transcribing antisense or double-stranded RNAs and three by mutated copies for suppressed expression of undesirable genes (the third strategy). By the first and the third strategies, hundreds of transgenic events and thousands of varieties with significant improvement of resistance to herbicides and pesticides, as well as nutritional quality, have been developed and approved for commercial release. Their application has significantly decreased the use of synthetic pesticides and the cost of crop production and increased the yield of crops and the benefits to farmers. However, almost all the events overexpressing endogenous genes remain at the testing stage, except one for fertility restoration and another for pyramiding herbicide tolerance. The novel functions conferred by the heterologously expressing exogenous genes under the control of constitutive promoters are usually absent in the recipient crops themselves or perform in different pathways. However, the endogenous proteins encoded by the overexpressing endogenous genes are regulated in complex networks with functionally redundant and replaceable pathways and are difficult to confer the desirable phenotypes significantly. It is concluded that heterologous expression of exogenous genes and suppressed expression by RNA interference and clustered regularly interspaced short palindromic repeats-cas (CRISPR/Cas) of undesirable genes are superior to the overexpression of endogenous genes for transgenic improvement of crops.

Characterization of phenylalanine ammonia-lyase genes facilitating flavonoid biosynthesis from two species of medicinal plant Anoectochilus.

Background: Anoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites. Methods: The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR). Results: AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis. Discussion: The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.