Expression In Transgenic Arabidopsis Research Articles

A shorter inverted repeat length in a hairpin RNA results in a stronger silencing efficacy

RNA interference (RNAi), particularly hairpin RNA (hpRNA) constructs, is a potent genetic manipulation tool, influencing traits that are crucial for crop improvement. Typical hpRNA constructs consist of inverted repeat sequences derived from the mRNA of the target gene, separated by a non-complementary spacer. This configuration initiates the formation of a double-stranded RNA structure due to the complementary nature of sense and antisense sequences in the transcribed RNA. In this research, two hpRNA constructs, namely hp-TFL1-S and hp-TFL1-L, with distinct lengths of inverted repeats, were designed to target TFL1, a pivotal regulator of both flowering timing and inflorescence structure. hp-TFL1-L construct encompasses all the 150 nucleotide sequences of the hp-TFL1-S construct and an extra 135 nucleotides. These constructs included a chalcone synthase intron as a spacer, resulting in hairpin double-stranded RNA molecules of 150 and 285 base pair after transcription. The PCR and qRT-PCR analysis results confirmed the accurate design of both constructs and their successful suppression of TFL1 mRNA expression in transgenic Arabidopsis. Interestingly, Arabidopsis lines carrying hp-TFL1-S and hp-TFL1-L exhibited distinct phenotypic features. hp-TFL1-S induced more pronounced phenotypic changes, resulting in earlier flowering compared to the hp-TFL1-L and control group. Moreover, hp-TFL1-S had a significant impact on inflorescence architecture, distinguishing it from both hp-TFL1-L and control plants. This study provides an example where inverted repeat length alters the efficacy of silencing, and in this case, surprisingly the shorter construct appears to have a stronger silencing efficacy. This argues multiple sized hpRNAs should be tested in RNAi-based gene silencing experiments.

Molecular mechanism that underlies cotton response to salt and drought stress revealed by complementary transcriptomic and iTRAQ analyses

Drought and salinity severely retard cotton growth and development, resulting in a substantial reduction in global cotton production. To investigate the molecular mechanisms underlying plant response, we compared the transcriptomic and proteomic expression profiles of cotton seedlings exposed to drought or salt stress using RNA-sequencing and iTRAQ sequencing, respectively, to gain a comprehensive understanding of the common and specific responses of cotton plants to salt and drought stress. The complexity of transcriptional and post-transcriptional regulations of salt and drought stress-responsive mechanisms was manifested most obviously by the tenuous yet intertwined relationships between the number of transcripts and the profile of protein expression. Intron retention was found to be the most prevalent alternative splicing type, suggesting a significant regulatory function at the transcriptional level. Enrichment analysis revealed that the majority of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were involved in the MAPK signaling pathway, phenylpropanoid biosynthesis, plant hormone signal transduction, and flavonoid biosynthesis. In particular, a number of MAPK cascade genes, including GhMEKK36, GhMAPKK3, GhMAPKK4, GhMAPK1, and GhMAPK5 were prominently upregulated by both the drought and salt stress treatments, and the functional role of these genes was investigated by ectopic expression in transgenic Arabidopsis, which exhibited significantly enhanced stress tolerance. This study advances our understanding of the molecular mechanisms that underpin cotton responses to drought and salt stress, offering crucial insights for further elaboration of the genes and pathways that are involved in cotton drought and salt tolerance.

Identification and Functional Characterization of the RcFAH12 Promoter from Castor Bean in Arabidopsis thaliana

Castor (Ricinus communis L.) seed oil is the commercial source of ricinoleate, a valuable raw material used in many industries. Oleoyl-12-hydroxylase (RcFAH12) is a key enzyme in the biosynthesis of ricinoleate, accumulating nearly 90% of the triacylglycerol in castor seeds. Little is known about the transcriptional regulation of RcFAH12. We used rapid amplification of cDNA 5′ ends (5′RACE) to locate the transcription start site (TSS) of RcFAH12, and the sequence of a 2605 bp region, −2506~+99, surrounding the TSS was cloned. We then investigated these regions to promote β-glucuronidase (GUS) expression in transgenic Arabidopsis by the progressive 5′ and 3′ deletions strategies. The GUS staining showed that the GUS accumulation varied in tissues under the control of different deleted fragments of RcFAH12. In addition, the GUS expression driven by the RcFAH12 promoter markedly accumulated in transgenic seeds, which indicated that RcFAH12 might play an important role in the biosynthesis of ricinoleic acid. This study will lay a potential foundation for developing a tissue-specific promoter in oil-seed crops.

Opposite Beet Cyst Nematode Infection Phenotypes of Transgenic Arabidopsis Between Overexpressing GmSNAP18 and AtSNAP2 and Between Overexpressing GmSHMT08 and AtSHMT4.

The rhg1-a GmSNAP18 (an α-SNAP) and Rhg4 GmSHMT08 are two major cloned genes conferring soybean cyst nematode resistance in Peking-type soybeans, but the application of α-SNAPs and SHMTs in cyst nematode management remains elusive. In this study, GmSNAP18 and GmSHMT08, together with their orthologs in Arabidopsis, AtSNAP2 (an α-SNAP) and AtSHMT4, were individually transformed into Arabidopsis Col-0 to generate the transgenic lines, and the growth of transgenic plants, beet cyst nematode (BCN) infection phenotypes, and AtSNAP2, AtSHMT4, and AtPR1 expression patterns were analyzed using Arabidopsis-BCN compatible interaction system, in addition with protein-protein interaction assay. Pulldown and BiFC assays revealed that GmSNAP18 and GmSHMT08 interacted with AtSHMT4 and AtSNAP2, respectively. Plant root growth was not impacted by overexpression of GmSNAP18 and AtSNAP2. However, overexpression of GmSHMT08 and AtSHMT4 both increased plant height, additionally, overexpression of GmSHMT08 decreased rosette leaf size. Overexpression of GmSNAP18 and GmSHMT08 both suppressed AtPR1 expression and significantly enhanced BCN susceptibility, while overexpression of AtSNAP2 and AtSHMT4 both substantially boosted AtPR1 expression and remarkably enhanced BCN resistance, in transgenic Arabidopsis. Overexpression of GmSNAP18 reduced, while overexpression of AtSNAP2 unaltered AtSHMT4 expression. Overexpression of GmSHMT08 and AtSHMT4 both suppressed AtSNAP2 expression in transgenic Arabidopsis. Thus, different expression patterns of AtPR1 and AtSHMT4 are likely associated with opposite BCN infection phenotypes of Arabidopsis between overexpressing GmSNAP18 and AtSNAP2, and between overexpressing GmSHMT08 and AtSHMT4; and boosted AtPR1 expression are required for enhanced BCN resistance in Arabidopsis. All these results establish a basis for extension of α-SNAPs and SHMTs in cyst nematode management.

Comparative analysis of various root active promoters by evaluation of GUS expression in transgenic Arabidopsis.

To prepare various root active promoters for expressing transgenes and prevent gene silencing caused by the repeated use of the same promoter, the expression characteristics of various root active promoters were comparatively evaluated using GUS as a reporter gene. The high-affinity potassium transporter (HKT1;1), the Shaker family potassium ion channel (SKOR), the Shaker family inward rectifying potassium channel (AKT1), the major facilitator superfamily protein (MFS1), and the senescence associated gene 14 (SAG14) promoter from Arabidopsis (Arabidopsis thaliana) were used, and for comparison, four additional constitutive or green tissue specific promoters in the expression vectors were also employed. As the Gateway cloning technology provided by Invitrogen can offer high efficiency and cloning reliability, and easy manipulation of fusion constructs in vitro, our expression vectors are based on binary (destination) vectors compatible with this cloning technique. These destination vectors are also advantageous for stable expression of the transgene, as the heat shock protein terminator is utilized. The AtHKT1;1, SKOR, AKT1, MFS1 and SAG14 promoters were all active in roots but showed slightly different tissue specificities: AtHKT1;1, SKOR, and MFS1 were dominantly active in vascular bundle tissue, while AtHKT1;1 and MFS1- but not SKOR, AKT1, and SAG14-were active in root tips. SKOR showed the strongest root-specificity, and SAG14 showed the highest activity among the five root active promoters. The activity of MFS was developmentally regulated. These destination vectors are now available to express multiple transgenes in transgenic plants, especially in roots.

Multi-omic dissection of the drought resistance traits of soybean landrace LX.

With diverse genetic backgrounds, soybean landraces are valuable resource for breeding programs. Herein, we apply multi-omic approaches to extensively characterize the molecular basis of drought tolerance in the soybean landrace LX. Initial screens established that LX performed better with PEG6000 treatment than control cultivars. LX germinated better than William 82 under drought conditions and accumulated more anthocyanin and flavonoids. Untargeted mass spectrometry in combination with transcriptomic analyses revealed the chemical diversity and genetic basis underlying the overall performance of LX landrace. Under control and drought conditions, significant differences in the expression of a suite of secondary metabolism genes, particularly those involved in the general phenylpropanoid pathway and flavonoid but not lignin biosynthesis, were seen in LX and William 82. The expression of these genes correlated with the corresponding metabolites in LX plants. Further correlation analysis between metabolites and transcripts identified pathway structural genes and transcription factors likely are responsible for the LX agronomic traits. The activities of some key biosynthetic genes or regulators were confirmed through heterologous expression in transgenic Arabidopsis and hairy root transformation in soybean. We propose a regulatory mechanism based on flavonoid secondary metabolism and adaptive traits of this landrace which could be of relevance to cultivated soybean.

Perception of the pathogen-induced peptide RGF7 by the receptor-like kinases RGI4 and RGI5 triggers innate immunity in Arabidopsis thaliana.

Signaling peptides play crucial roles in plant growth, development and defense. We report here that the Arabidopsis thaliana secreted peptide, ROOT MERISTEM GROWTH FACTOR 7 (RGF7), functions as an endogenous elicitor to trigger plant immunity. Expression of the RGF7 precursor-encoding gene (preRGF7) is highly induced in Arabidopsis leaves upon infection by the bacterial pathogen Pseudomonas syringae. The pathogen-responsive preRGF7 expression is regulated by the transcription factor WRKY33 and its upstream mitogen-activated protein kinases MPK3/MPK6 and calcium-dependent protein kinases CPK5/CPK6. In the absence of pathogen attack, chemically induced expression of preRGF7 in transgenic Arabidopsis plants was sufficient to trigger immune responses. Pre-induction of preRGF7 expression in transgenic Arabidopsis also led to enhanced immune responses and increased resistance to P.syringae infection. Biochemical and genetic analyses demonstrated that RGF7 is perceived by the leaf-expressed RGF1 INSENSITIVE (RGI) family receptors RGI4 and RGI5. The SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) BAK1 and SERK4 are also involved in RGF7 perception via forming RGF7-induced receptor-complexes with RGI4 and RGI5. These results indicate that the pathogen-induced RGF7 peptide, perceived by the RGI4/RGI5-BAK1/SERK4 receptor complexes, acts as a new damage-associated molecular pattern (DAMP) and plays a significant role in Arabidopsis immunity.

A celery transcriptional repressor AgERF8 negatively modulates abscisic acid and salt tolerance.

Ethylene response factors (ERFs) widely exist in plants and have been reported to be an important regulator of plant abiotic stress. Celery, a common economic vegetable of Apiaceae, contains lots of ERF transcription factors (TFs) with various functions. AP2/ERF TFs play positive or negative roles in plant growth and stress response. Here, AgERF8, a gene encoding EAR-type AP2/ERF TF, was identified. The AgERF8 mRNA accumulated in response to both abscisic acid (ABA) signaling and salt treatment. AgERF8 wasprovingtobe a nucleus-located protein and could bind to GCC-box. The overexpression of AgERF8 in Arabidopsis repressed the transcription of downstream genes, AtBGL and AtBCH. Arabidopsis overexpressing AgERF8 gene showed inhibited root growth under ABA and NaCl treatments. AgERF8 transgenic lines showed low tolerance to ABA and salt stress than wild-type plants. Low increment in SOD and POD activities, increased accumulation of MDA, and significantly decreased plant fresh weights and chlorophyll levels were detected in AgERF8 hosting lines after treated with ABA and NaCl. Furthermore, the overexpression of AgERF8 also inhibited the levels of ascorbic acid and antioxidant-related genes (AtCAT1, AtSOD1, AtPOD, AtSOS1, AtAPX1, and AtP5CS1) expression in transgenic Arabidopsis. This finding indicated that AgERF8 negatively affected the resistance of transgenic Arabidopsis to ABA and salt stress through regulating downstream genes expression and relevant physiological changes. It will provide a potential sight to further understand the functions of ERF TFs in celery.

Genetic variation in ZmTIP1 contributes to root hair elongation and drought tolerance in maize.

SummaryDrought is a major abiotic stress that threatens maize production globally. A previous genome‐wide association study identified a significant association between the natural variation of ZmTIP1 and the drought tolerance of maize seedlings. Here, we report on comprehensive genetic and functional analysis, indicating that ZmTIP1, which encodes a functional S‐acyltransferase, plays a positive role in regulating the length of root hairs and the level of drought tolerance in maize. We show that enhancing ZmTIP1 expression in transgenic Arabidopsis and maize increased root hair length, as well as plant tolerance to water deficit. In contrast, ZmTIP1 transposon‐insertional mutants displayed the opposite phenotype. A calcium‐dependent protein kinase, ZmCPK9, was identified as a substrate protein of ZmTIP1, and ZmTIP1‐mediated palmitoylation of two cysteine residues facilitated the ZmCPK9 PM association. The results of this research enrich our knowledge about ZmTIP1‐mediated protein S‐acylation modifications in relation to the regulation of root hair elongation and drought tolerance. Additionally, the identification of a favourable allele of ZmTIP1 also provides a valuable genetic resource or selection target for the genetic improvement of maize.

A Neighboring Aromatic-Aromatic Amino Acid Combination Governs Activity Divergence between Tomato Phytoene Synthases.

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue-specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Targeted Transcriptional Activation in Plants Using a Potent Dead Cas9-Derived Synthetic Gene Activator.

Genetic tools for specific perturbation of endogenous gene expression are highly desirable for interrogation of plant gene functions and improvement of crop traits. Synthetic transcriptional activators derived from the CRISPR/Cas9 system are emerging as powerful new tools for activating the endogenous expression of genes of interest in plants. These synthetic constructs, generated by tethering transcriptional activation domains to a nuclease-dead Cas9 (dCas9), can be directed to the promoters of endogenous target genes by single guide RNAs (sgRNAs) to activate transcription. Here, we provide a detailed protocol for targeted transcriptional activation in plants using a recently developed, highly potent dCas9 gene activator construct referred to as dCas9-TV. This protocol covers selection of sgRNA targets, construction of sgRNA expression cassettes, and screening for an optimal sgRNA using a protoplast-based promoter-luciferase assay. Finally, the dCas9-TV gene activator coupled with the optimal sgRNA is delivered into plants via Agrobacterium-mediated transformation, thereby enabling robust upregulation of target gene expression in transgenic Arabidopsis and rice plants. © 2019 by John Wiley & Sons, Inc.

Expression of cotton PLATZ1 in transgenic Arabidopsis reduces sensitivity to osmotic and salt stress for germination and seedling establishment associated with modification of the abscisic acid, gibberellin, and ethylene signalling pathways

BackgroundZinc-finger transcription factors play central roles in plant growth, development and abiotic stress responses. PLATZ encodes a class of plant-specific zinc-finger transcription factor. However, biological functions or physiological mechanism controlled by PLATZ are currently limited.ResultsGhPLATZ1 transcripts were considerably up-regulated by NaCl, mannitol, abscisic acid (ABA) and gibberellin (GA) treatments. Transgenic Arabidopsis by ectopic expression of GhPLATZ1 exhibited faster seed germination and higher seedling establishment under salt and mannitol stresses than those of wild type (WT), indicating enhanced osmotic insensitivity in GhPLATZ1 transgenic Arabidopsis. The ABA content in dry seeds of GhPLATZ1 transgenic Arabidopsis was lower than that of WT whereas the ABA content was not changed in germinating seeds under salt stress. Seed germination was faster than but the seedling establishment of transgenic Arabidopsis was similar to WT. Besides, GhPLATZ1 transgenic and WT Arabidopsis exhibited insensitivity to paclobutrazol (PAC), a GA biosynthesis inhibitor, whereas exogenous GA could eliminate the growth difference between GhPLATZ1 transgenic and WT Arabidopsis under salt stress. Moreover, exogenous 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, exerted similar effects to GA. Furthermore, ABI4 and ETO1 transcripts were significantly down-regulated, whereas ACS8 was up-regulated in GhPLATZ1 transgenic Arabidopsis under salt stress.ConclusionsIn conclusion, GhPLATZ1 had broad influence in responses to salt and mannitol stresses in transgenic Arabidopsis during seed germination and seedling establishment. The effect of GhPLATZ1 expression in transgenic Arabidopsis might be mediated by the ABA, GA, and ethylene pathways. Thus, this study provided new insights into the regulatory network in response to abiotic stresses in plants.

The Glycine soja NAC transcription factor GsNAC019 mediates the regulation of plant alkaline tolerance and ABA sensitivity

Overexpression of Gshdz4 or GsNAC019 enhanced alkaline tolerance in transgenic Arabidopsis. We proved that Gshdz4 up-regulated both GsNAC019 and GsRD29B but GsNAC019 may repress the GsRD29B expression under alkaline stress. Wild soybean (Glycine soja) has a high tolerance to environmental challenges. It is a model species for dissecting the molecular mechanisms of salt-alkaline stresses. Although many NAC transcription factors play important roles in response to multiple abiotic stresses, such as salt, osmotic and cold, their mode of action in alkaline stress resistance is largely unknown. In our study, we identified a G. soja NAC gene, GsNAC019, which is a homolog of the Arabidopsis AtNAC019 gene. GsNAC019 was highly up-regulated by 50mM NaHCO3 treatment in the roots of wild soybean. Further investigation showed that a well-characterized transcription factor, Gshdz4 protein, bound the cis-acting element sequences (CAATA/TA), which are located in the promoter of the AtNAC019/GsNAC019 genes. Overexpression of Gshdz4 positively regulated AtNAC019 expression in transgenic Arabidopsis, implying that AtNAC019/GsNAC019 may be the target genes of Gshdz4. GsNAC019 was demonstrated to be a nuclear-localized protein in onion epidermal cells and possessed transactivation activity in yeast cells. Moreover, overexpression of GsNAC019 in Arabidopsis resulted in enhanced tolerance to alkaline stress at the seedling and mature stages, but reduced ABA sensitivity. The closest Arabidopsis homolog mutant plants of Gshdz4, GsNAC019 and GsRD29B containing athb40, atnac019 and atrd29b were sensitive to alkaline stress. Overexpression or the closest Arabidopsis homolog mutant plants of the GsNAC019 gene in Arabidopsis positively or negatively regulated the expression of stress-related genes, such as AHA2, RD29A/B and KIN1. Moreover, this mutation could phenotypically promoted or compromised plant growth under alkaline stress, implying that GsNAC019 may contribute to alkaline stress tolerance via the ABA signal transduction pathway and regulate expression of the downstream stress-related genes.

High Constitutive Overexpression of Glycosyl Hydrolase Family 17 Delays Floral Transition by Enhancing FLC Expression in Transgenic Arabidopsis.

Vitis vinifera glycosyl hydrolase family 17 (VvGHF17) is a grape apoplasmic β-1,3-glucanase, which belongs to glycosyl hydrolase family 17 in grapevines. β-1,3-glucanase is not only involved in plant defense response but also has various physiological functions in plants. Although VvGHF17 expression is negatively related to the length of inflorescence in grapevines, the physiological functions of VvGHF17 are still uncertain. To clarify the physiological functions of VvGHF17, we conducted a phenotypic analysis of VvGHF17-overexpressing Arabidopsis plants. VvGHF17-overexpressing Arabidopsis plants showed short inflorescence, similar to grapevines. These results suggested that VvGHF17 might negatively regulate the length of inflorescence in plants. VvGHF17 expression induced a delay of floral transition in Arabidopsis plants. The expression level of FLOWERING LOCUS C (FLC), known as a floral repressor gene, in inflorescence meristem of transgenic plants were increased by approximately 10-fold as compared with wild plants. These results suggest that VvGHF17 induces a delay of floral transition by enhancing FLC expression and concomitantly decreases the length of plant inflorescence.

The promoter of fatty acid desaturase on chromosome C5 in Brassica napus drives high-level expression in seeds

The gene fatty acid desaturase 2 (FAD2) exists in multiple copies in the Brassica napus genome and encodes an enzyme that catalyzes the conversion of oleic acid to linoleic acid. In the present study, we characterized the regulatory region controlling the expression of an FAD2 gene located on chromosome C5 of Brassica napus and named it BnFAD2-C5. A long intron was found within the 5′-untranslated region (5′-UTR) of the BnFAD2-C5 gene. This intron, compared with an intron-less control, conferred up to a sixfold increase in green fluorescent protein (GFP) expression in transgenic Arabidopsis, thus suggesting that it makes function through intron-mediated enhancement. The sequence containing the promoter and intron was identified to promote high levels of gene expression in genital organs, particularly in seeds, using qRT-PCR and transgenic Arabidopsis. We identified the different promoter regions responsible for the tissue-specific gene expression through a deletion analysis of the BnFAD2-C5 promoter and a β-glucuronidase and GFP reporter system. The results showed that the −1020 to −319 bp region primarily controls BnFAD2-C5 gene expression in the root, whereas the −1020 to −581 bp region controls expression in the stem, the −581 to −319 bp region controls expression in the leaf, and the −1257 to −1020 bp region probably controls expression in the floral parts. The −319 to −1 bp region is also important, conferring high-level transcription in the seeds. The transcription of BnFAD2-C5 could be induced by salicylic acid and jasmonic acid, and the relative response elements were identified in the −1257 to −1020 bp region and −319 to −1 bp region, respectively.

The promoter from the Citrus unshiu carotenoid isomerase gene directs differential GUS expression in transgenic Arabidopsis

Carotenoid isomerase (CRTISO) catalyzes the isomerization of prolycopene to all-trans-lycopene in the carotenoid biosynthetic pathway. We isolated a promoter region of CuCRTISO from Citrus unshiu. We determined whether the promoter encoded organ-specific or developmental-specific expression and identified possible cis-acting promoter elements. The promoter and two truncated versions were fused to the β-glucuronidase (GUS) gene and transformed into Arabidopsis thaliana. Transgenic lines expressing the 2501-bp promoter (pCiso-Prom1) and truncated promoters (pCiso-Prom2 and pCiso-Prom3) showed the same developmental and organ-specific activity. GUS expression was detected in the cotyledon and root at 5 and 10 days after germination, mature leaf, and anther. The CuCRTISO promoter contained several cis-acting elements involved in hormonal and environmental stress. Drought stress or abscisic acid treatment did not induce GUS expression in any transgenic lines. Heat stress induced GUS expression in the pCiso-Prom1 line; this promoter construct contains the heat stress-responsive element. Ethylene and cold stress treatments induced GUS expression only in the pCiso-Prom3 line, although all transgenic lines contained the same cis-acting ethylene and low-temperature response elements, which could indicate the existence of unknown repressor element(s) in the CuCRTISO promoter. The CuCRTISO expression of Citrus unshiu during fruit maturation showed that in the green stage, it was highly expressed and then subsequently decreased after the stage. These studies indicate that CuCRTISO promoter activity is regulated in a developmental and organ-specific manner that responds to heat, cold, and ethylene. These results provide new insights into the role of cis-acting element(s) in CuCRTISO promoter activity.

GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots.

BackgroundPromoters play important roles in gene expression and function. There are three basic types of promoters: constitutive, specific, and inducible. Constitutive promoters are widely used in genetic engineering, but these promoters have limitations. Inducible promoters are activated by specific inducers. Tissue-specific promoters are a type of specific promoters that drive gene expression in specific tissues or organs. Here, we cloned and characterized the GmPRP2 promoter from soybean. The expression pattern indicated that this promoter is root-preferential in transgenic Arabidopsis and the hairy roots of soybean. It can be used to improve the root resistance or tolerance to pathogens, pests, malnutrition and other abiotic stresses which cause extensive annual losses in soybean production.ResultsThe GmPRP2 promoter (GmPRP2p-1062) was isolated from soybean cv. Williams 82. Sequence analysis revealed that this promoter contains many cis-acting elements, including root-specific motifs. The GmPRP2p-1062 and its 5’-deletion fragments were fused with the GUS reporter gene and introduced into Arabidopsis and the hairy roots of soybean to further determine promoter activity. Histochemical analysis in transgenic Arabidopsis showed that GUS activity was mainly detected in roots and hypocotyls in all deletion fragments except GmPRP2p-471 (a 5’-deletion fragment of GmPRP2p-1062 with 471 bp length). GUS activity was higher in transgenic Arabidopsis and hairy roots with GmPRP2p-1062 and GmPRP2p-852 (a 5’-deletion fragment of GmPRP2p-1062 with 852 bp length) constructs than the other two constructs. GUS activity was enhanced by NaCl, PEG, IAA and JM treatments and decreased by SA, ABA and GA treatments in transgenic Arabidopsis.ConclusionsGmPRP2p-1062 is a root-preferential promoter, and its core fragment for root-preferential expression might lie between −369 and +1. GmPRP2p-852 may be useful in the genetic engineering of novel soybean cultivars in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0245-z) contains supplementary material, which is available to authorized users.

Sorghum Extracellular Leucine-Rich Repeat Protein SbLRR2 Mediates Lead Tolerance in Transgenic Arabidopsis

A sorghum pathogen-inducible gene predicted to encode a simple extracellular leucine-rich repeat (LRR) protein SbLRR2 was previously isolated. LRR was the only domain identified in SbLRR2 and its homologous sequences. Phylogenetic analysis revealed that they are distinct from the simple extracellular LRR proteins reported previously. Agrobacterium-mediated transient expression in tobacco leaf cells demonstrated that the SbLRR2-EYFP (enhanced yellow fluorescent protein) fusion protein was targeted to the extracellular space. Transgenic analysis of SbLRR2 revealed its role in enhancing lead [Pb(II)] tolerance in Arabidopsis. Consequently, SbLRR2-overexpressing lines were found to show alleviated Pb(II)-induced root inhibition, lower levels of Pb(II) accumulation and enhanced transcription of AtPDR12 which encodes a plasma membrane ATP-bind cassette (ABC)-type transporter formerly shown to contribute to Pb(II) detoxification. However, all the Pb(II) tolerance responses were abolished when SbLRR2 was overexpressed in an atpdr12 T-DNA insertion line. The extracellular localization of SbLRR2 was also shown to be essential for the Pb(II) phenotypes and AtPDR12 up-regulation. Taken together, SbLRR2 appears to mediate Pb(II) tolerance through the elevation of AtPDR12 expression in transgenic Arabidopsis, thus activating a glutathione-independent mechanism for detoxification. Further investigations revealed the Pb(II)-induced transcriptional activation of SbLRR2 and several highly conserved AtPDR12 homologs in sorghum seedlings, suggesting the possibility of a common molecular mechanism for Pb(II) tolerance in diverse plant species.

A RING‐type E3 ligase controls anther dehiscence by activating the jasmonate biosynthetic pathway gene DEFECTIVE IN ANTHER DEHISCENCE1 in Arabidopsis

Suppression of expression of DAF [DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1)-Activating Factor], a gene that encodes a putative RING-finger E3 ligase protein, causes non-dehiscence of the anthers, alters pollen development and causes sterility in 35S:DAF RNAi/antisense Arabidopsis plants. This mutant phenotype correlates with the suppression of DAF but not with expression of the two most closely related genes, DAFL1/2. The expression of DAD1 was significantly reduced in 35S:DAF RNAi/antisense plants, and complementation with 35S:DAF did not rescue the dad1 mutant, indicating that DAF acts upstream of DAD1 in jasmonic acid biosynthesis. This assumption is supported by the finding that 35S:DAF RNAi/antisense plants showed a similar cellular basis for anther dehiscence to that found in dad1 mutants, and that external application of jasmonic acid rescued the anther non-dehiscence and pollen defects in 35S:DAF antisense flowers. We further demonstrate that DAF is an E3 ubiquitin ligase and that its activity is abolished by C132S and H137Y mutations in its RING motif. Furthermore, ectopic expression of the dominant-negative C132S or H137Y mutations causes similar indehiscence of anthers and reduction in DAD1 expression in transgenic Arabidopsis. This result not only confirms that DAF controls anther dehiscence by positively regulating the expression of DAD1 in the jasmonic acid biosynthesis pathway, but also supports the notion that DAF functions as an E3 ubiquitin ligase, and that the conserved RING-finger region is required for its activity.

A positive regulatory role of the watermelon ClWRKY70 gene for disease resistance in transgenic Arabidopsis thaliana

A pathogen-inducible WRKY cDNA was cloned from the leaves of watermelon (Citrullus lanatus) seedlings 24 h after inoculation with Cladosporium cucumerinum. The deduced protein of the gene, designated as ClWRKY70, was classified as a group III WRKY protein based on its single WRKY domain containing a Cys2HisCys zinc-finger motif. Its Arabidopsis thaliana sequence homologue (AtWRKY70) has been described as playing an important role in the plant defense response. ClWRKY70 gene transcripts were highly accumulated in watermelon by salicylic acid treatment, but not by jasmonic acid. By evaluating target gene expression in transgenic Arabidopsis overexpressing the ClWRKY70 gene, it is suggested that the watermelon WRKY gene may play a positive regulatory role in plant resistance against pathogen attack.