Light-responsive Elements Research Articles

Functional Identification of EjGIF1 in Arabidopsis and Preliminary Analysis of Its Regulatory Mechanisms in the Formation of Triploid Loquat Leaf Heterosis.

Although several results have been obtained in triploid loquat heterosis (i.e., leaf size of triploid loquat) studies in the past years, the underlying mechanisms of the heterosis are still largely unknown, especially the regulation effects of one specific gene on the corresponding morphology heterosis. In this study, we sought to further illustrate the regulatory mechanisms of one specific gene on the leaf size heterosis of triploid loquats. A leaf size development-related gene (EjGIF1) and its promoter were successfully cloned. Ectopic expression of EjGIF1 in Arabidopsis showed that the leaf size of transgenic plantlets was larger than that of WTs, and the transgenic plantlets had more leaves than WTs. Quantitative Reverse Transcription PCR (qRT-PCR) showed that the expression level of EjGIF1 showed an AHP expression pattern in most of the hybrids, and this was consistent with our previous phenotype observations. Structure analysis of EjGIF1 promoter showed that there were significantly more light-responsive elements than other elements. To further ascertain the regulatory mechanisms of EjGIF1 on triploid loquat heterosis, the methylation levels of EjGIF1 promoter in different ploidy loquats were analyzed by using bisulfite sequencing. Surprisingly, the total methylation levels of EjGIF1 promoter in triploid showed a decreasing trend compared with the mid-parent value (MPV), and this was also consistent with the qRT-PCR results of EjGIF1. Taken together, our results suggested that EjGIF1 played an important role in promoting leaf size development of loquat, and demethylation of EjGIF1 promoter in triploid loquats caused EjGIF1 to exhibit over-dominance expression pattern and then further to promote leaf heterosis formation. In conclusion, EjGIF1 played an important role in the formation of triploid loquat leaf size heterosis.

Genome-wide analysis and characterization of GRAS family in switchgrass

ABSTRACT Panicum virgatum, a model plant of cellulosic ethanol conversion, not only has high large biomass and strong adaptability to soil, but also grows well in marginal soil and has the advantage of improving saline-alkali soil. GRAS transcription factor gene family play important roles in individual environment adaption, and these vital functions has been proved in several plants, however, the research of GRAS in the development of switchgrass (Panicum virgatum) were limited. A comprehensive study was investigated to explore the relationship between GRAS gene family and resistance. According to the phylogenetic analysis, a total of 144 GRAS genes were identified and renamed which were classified into eight subfamilies. Chromosome distribution, tandem and segmental repeats analysis indicated that gene duplication events contributed a lot to the expansion of GRAS genes in the switchgrass genome. Sixty-six GRAS genes in switchgrass were identified as having orthologous genes with rice through gene duplication analysis. Most of these GRAS genes contained zero or one intron, and closely related genes in evolution shared similar motif composition. Interaction networks were analyzed including DELLA and ten interaction proteins that were primarily involved in gibberellin acid mediated signaling. Notably, online analysis indicated that the promoter regions of the identified PvGRAS genes contained many cis-elements including light responsive elements, suggesting that PvGRAS might involve in light signal cross-talking. This work provides key insights into resistance and bioavailability in switchgrass and would be helpful to further study the function of GRAS and GRAS-mediated signal transduction pathways.

Characterization, expression and function analysis of anthocyanidin reductase gene from Vitis vinifera L. cv. Cabernet Sauvignon under UV-C irradiation

Flavonoids are important polyphenols, and widely distributed in plants. Anthocyanidin reductase (ANR), a precursor enzyme in (-)- epicatechin synthesis, could play an important role in anthocyanins biosynthesis. In the present study, the ANR gene was cloned from Vitis vinifera L. cv. Cabernet Sauvignon. Compared with VvANR (GenBank Accession No. NM_001280956.1), they differed in only 4 bp and shared similarity of 99.6%. The ANR protein belongs to the cl33424 Superfamily. In addition, many light-responsive elements and abiotic stress-related elements (ABRE, MYB, WRKY, etc.) were found in the promoter region of the ANR gene. STRING analysis predicted that VvMYBPA1 and VvMYBPAR may be involved in regulating the expression of the ANR gene. Different UV-C irradiation times were used for Cabernet Sauvignon berries during grape berry development, and the comparison analysis showed that UV-C irradiation improved significantly the accumulation of catechin and epicatechin during 20–50 days after full bloom, especially, under UV-C irradiation for 5 min. Furthermore, UV-C irradiation for 5 min had a greater effect on the expression of the ANR gene, the accumulation of catechin and epicatechin, and the enzymatic activity of ANR than UV-C irradiation for 10 min. Through the virus-induced gene silencing system, the ANR-silenced grape leaves were chlorotic, in which the contents of catechin and epicatechin were both significantly lower than those in the control, showing a very significant difference, respectively. In summary, our results demonstrated that the ANR gene played a crucial role in the accumulation of epicatechin in grapes, and its expression was upregulated by UV-C irradiation.

Isolation, Cloning and Characterization of a Constitutive Plant from Potato Aquaporin Gene

Plasma membrane intrinsic proteins (PIP1) are the most common integral membrane proteins belong to a larger family of intrinsic aquaporin proteins. They are member of aquaporin gene family and have gained importance as highly expressed genes in plants. In this study, the promoter of aquaporin PIP1 gene was identified, analyzed and retrieved from high throughput genomic sequence (HTGS) database. The cis-acting regulatory elements, transcription start sites and transcription factor binding sites of selected promoter were identified through different bio-informatics tools. Many light responsive, phytohormone, stress and defense related cis-regulatory elements were detected in PIP1 promoter region indicating its role as a constitutive promoter. The PIP1 promoter was isolated from Solanum tuberosum. It was initially cloned in TA vector (pTZ57R/T) and later transferred to plant expression binary vectors, pGR1 and pGA482 for transient and stable expression studies in tobacco. The GUS expression results of PIP1 promoter in different tobacco tissues showed its functional importance in regulating gene expression in a constitutive manner. Further, it was concluded that the PIP1 aquaporin promoter is constitutively expressed with a strength equivalent to CaMV 2x35S promoter. These findings indicated the significance of isolated promoter for genetic engineering of plants for crop improvement.

Identification of SABATH Family Members in Solanum lycopersicum and Their Expression Patterns Under Abiotic/Biotic Stresses

The SABATH family proteins catalyse S-adenosyl-l-methionine-dependent methylation of hormones, signal molecules and other metabolites in plants, and they play an important role in various processes of plant development, especially in plant resistance. Solanum lycopersicum is an important model plant and provides fleshy fruits that are widely used. To date, the SABATH family has not been investigated in Solanum lycopersicum. Here, a total of 20 SABATH amino acid sequences were identified and characterized in S. lycopersicum (SlSABATH). Phylogenetic analysis of different species indicated that these proteins could be classified into 3 groups, and proteins with similar functions were classified into the subclades. Chromosomal mapping showed that SlSABATH proteins were distributed on 7 chromosomes. Conserved domain and motif analysis revealed that these proteins shared homology in 10 motifs, and motifs 2 and 5 were the specific binding sites for S-adenosyl-l-methionine. The structural features of each SlSABATH member showed that those in the same subgroup containing the same exon-intron patterns generally catalysed the same substrate. There were 7 pairs in the SlSABATH family that were produced from the tandem duplication of genes and 2 pairs that were produced during the segmental duplication of genes; these were primary driving forces in the evolution of SlSABATH family. Plant hormone-responsive, stress-inducible and defence-related elements and light-responsive elements accounted for most of the cis regulatory-elements. Gene expression profiling revealed that most of the SlSABATH members were expressed in tissues, Solyc01g080970 was significantly expressed in fully opened flowers and Solyc09g091550 was abundantly expressed in stems. Many SlSABATH genes were significantly up- or downregulated by stress treatments (Pseudomonas syringae pv. tomato DC3000, cold and drought). The above results provide references to further study the function of SlSABATH family members in S. lycopersicum.

Characterization of two predicted DASH-related proteins from the green alga Volvox carteri provides new insights into their light-mediated transcript regulation and DNA repair activity

Volvocine lineage, categorized in the Chlorophyta phylum, includes multicellular algae (such as Volvox) and the single-celled Chlamydomonas. After genome sequencing and annotation of V. carteri, some predicted photoreceptor coding genes were discovered in its genome. These genes create a sophisticated light-sensing system to adjust the physiology, growth, development, and behavior of the green alga. The specific light-sensitive modules are used to engineer the optogenetic tools as photo-switchable instruments. Given this, the current study presents a description of two nuclear photolyase/cryptochrome gene loci (VcCPF5 and VcCPF8) in V. carteri. In this regard, we conducted in-silico and experimental analyses to investigate the protein functions. Phylogenetic analysis revealed that these genes encode two proteins related to DASH-cryptochrome subclade. In addition, the 3D model of these proteins showed their similarity with photolyase protein family members with DNA repair activity. Furthermore, VcCPF5 and VcCPF8 transcripts increased after red and blue-light treatments during the gonidia maturation phase of microalga which were confirmed by determining the light responsive elements in their putative promoters. Moreover, based on the spectrophotometric analysis of purified and un-purified proteins, they had absorption in the blue-light spectrum like the other members of the cryptochrome/photolyase protein family. Additionally, the transgenic Escherichia coli containing the heterologous tagged proteins were found which increase the survival capability under UV-irradiation compared to the control samples. In conclusion, our findings demonstrated that the expression of DASH-related genes in Volvox rely on the light quality. Moreover, in- vivo and in-silico studies confirmed that these proteins are involved in the light dependent DNA repair process. Then they can be suggested as potential candidates for designing Light-Inducible DNA-Repair (LIDR) tools in the optogenetics studies.

Identification and Characterization of DlGI Promoter Involved in Photoperiod, Light Intensity, Hormone, and DlELF4 Response from Longan

In fruit trees, flowering is a key event followed by fruit development and seed production. Gigentea (GI), a clock-associated gene, is known to contribute to photoperiodic flowering and circadian clock control in Arabidopsis thaliana. However, its functions in woody fruit trees remain unclear. In this study, a 2000 bp promoter fragment of the longan (Dimocarpous longan) DlGI gene was isolated from the genomic DNA of longan ‘Honghezi’ by polymerase chain reaction amplification. The DlGI promoter contained two main types of potential cis-acting elements: light-responsive and hormone-responsive elements. The promoter was fused with the β-glucuronidase (GUS) reporter gene of pBI121 to generate the pDlGI:GUS construct. GUS histochemical staining of transgenic A. thaliana revealed that DlGI might play a role in different developmental phases of longan. Exposure of transgenic A. thaliana to varying light intensities showed that the GUS activity increases with increased light intensity. Transient expression of pDlGI::GUS in Nicotiana benthamiana showed that the GUS activity was higher and reached peak a few hours earlier under short-day (SD) than long-day conditions. Exposure to different hormonal treatments revealed that the transcript level of GUS was activated by gibberellin (GA3) and indoleacetic acid (IAA) but suppressed by abscisic acid and methyl jasmonate treatment. In addition, N. benthamiana transient assay and dual-luciferase assay revealed that the presence of early flowering 4 (ELF4) homologs of longan (DlELF4-1 and DlELF4-2) significantly activated the DlGI promoter. The positive response of DlGI promoter to high light-intensity, SD photoperiod, GA3 and IAA signals, and DlELF4 transcription factor suggest that DlGI may function as a circadian clock and play a role in responding to SD conditions and other signals in flower initiation of longan.

Cloning and activity analysis of the promoter of nucleotide exchange factor gene ZjFes1 from the seagrasses Zostera japonica

After HSP70 binds to the J domain of the substrate and co-chaperone protein, ATP is hydrolyzed to ADP, and the nucleotide exchange factors (NEFs) promote the release of ADP. Under physiological conditions, the nucleotide exchange step is the rate-limiting step, which is accelerated by NEFs. In this study, the promoter of nucleotide exchange factor ZjFes1 was cloned, and its expression in tissues and under heat stress was studied to understand the regulatory mechanism of ZjFes1 and provide the molecular basis to study heat tolerance mechanism of seagrass. It was found that the promoter has common cis-acting elements in promoter and enhancer regions CAAT-box, as well as light response elements AE-box, Box 4 and TCCC-motif, a cis-acting regulatory element essential for the anaerobic induction of ARE, hormone response elements CGTCA-motif and TGACG-motif (MeJA response element), GARE-motif (gibberellin response element), TGA-element (auxin response element), a cis-acting regulatory element related to meristem expression CAT-box, and a cis-acting element involved in defense and stress responsiveness of TC-rich repeats. Two-week-old seedlings exhibited weak GUS activities in their cotyledons. In addition, the AtFes1A promoter was constitutively active in the anthers. After exposure to 38 °C for 2 h, the root tips of two-week-old seedlings were stained a strong blue. Heat-inducible activities of GUS were also observed in the cotyledons, roots, leaves, anthers, sepals and siliques.

The determination of a novel inducible WY172 promoter derived from Oidium heveae HO-73

In plant genetic engineering, to ensure the efficiently expression of foreign genes in recipient plant cells require high-quality promoters. In this study, a novel promoter, WY172, derived from Oidium heveae B.A. Steinm, was isolated and functionally characterized. GUS staining and GUS activity tests indicated that WY172 could effectively drive GUS expression in both monocotyledonous rice and dicotyledonous tobacco. The micro-hypersensitive response revealed that transgenic tobacco harbouring WY172 and HpaXm had more blue necrotic cells than that of positive control of CaMV35S-HpaXm transgenic tobacco. As well, TMV inoculation tests showed fewer lesions on the leaves of WY172-HpaXm transgenic tobacco than those of the positive control, indicating that WY172 could drive the expression of exogenous functional genes HpaXm. Furthermore, the expression level of the HpaXm gene in WY172-HpaXm transgenic tobacco after TMV inoculation was 7.1 times that of uninoculated tobacco. And WY172 also had a relative stronger ability to drive expression of HpaXm after TMV inoculation compared with non-inoculated plants, indicating that WY172 might be a pathogen-inducible promoter. Bioinformatics analysis indicated that WY172 is rich in pathogen-induced cis-acting elements such as auxin response element and light response elements, and induction experiments demonstrated that WY172 drives GUS expression after exogenous application of IAA, which both supported that WY172 might be an inducible promoter. In conclusion, WY172 promoter is an IAA- and pathogen-inducible promoter, which might has high potential for use in a wide range of hosts. By blasting and predicting the Oidium heveae B.A. Steinm genome, we obtained and preliminarily demonstrated that the WY172 promoter that may be induced by IAA and TMV. WY172 can play a role in rice and tobacco, and can drive efficient expression of HpaXm in transgenic tobacco and produce TMV resistance and micro-hypersensitive response.

Characterization of the leaf rust responsive ARF genes in wheat (Triticum aestivum L.).

Genome-wide identification, classification, functional characterization and expression analysis of Auxin Responsive Factor (ARF) gene family in wheat reveal their attributes and role during leaf rust infection. Auxins are important plant growth regulators that also impact plant-pathogen interaction. Auxin responsive factors (ARF) are plant specific transcription factors that control responses to auxins. Whole genome investigation of ARF gene family is limited in allohexaploid wheat (Triticum aestivum L.). Comprehensive study of this gene family was carried out by employing the currently available reference genome sequence of wheat. In total, 27 ARF genes were identified and located on the wheat genome as well as were positioned on wheat chromosome arms. Additionally, examination of the predicted genes unveiled a decent degree of relatedness within and among the phylogenetic clades. Leaf rust, caused by the obligate biotrophic fungal pathogen Puccinia triticina, is responsible for drastic loss of wheat crop worldwide reducing grain yield by 10-90%. Expression profiling of ARF genes in retort to leaf rust infection indicated their differential regulation during this plant-pathogen interaction. Highest expression of ARF genes were observed at 12 hpi that was maintained up to 72 hpi during incompatible interaction, whereas the high expression levels receded at 48 hpi during compatible interactions. Few of the identified ARF genes were likely to be post-transcriptionally regulated by microRNAs. Many light and stress responsive elements were detected in the promoter regions of ARF genes. Microsynteny analysis showed the conservation of ARF genes within the members of the Poaceae family. This study provides fundamental details for understanding the different types of ARF genes in wheat and there putative roles during leaf rust-wheat interaction.

Four-Dimensional Printed Liquid Crystalline Elastomer Actuators with Fast Photoinduced Mechanical Response toward Light-Driven Robotic Functions.

Remote light exposure of photoresponsive liquid crystalline polymers has drawn great attention over the last years as an attractive strategy to generate mechanical work with high spatial resolution. To tailor these materials into practical engineering devices, it is of key importance to gain control over their morphology and thus precisely program their mechanical response, which must also be fast and relevant in magnitude. In this communication, we report the four-dimensional (4D) printing of azobenzene-containing liquid crystalline elastomers (LCEs) that respond to light. During extrusion of the LCE precursor, mesogen orientation is defined by the needle's moving direction enabling a precise definition of the director, which is later fixed by photopolymerization. Fast mechanical responses have been observed in these 4D printed LCE elements when excited with ultraviolet (UV) light. These 4D printed elements lift objects many times heavier than their own weight, demonstrating a capacity to produce effective work. Photochemical and photothermal contributions to the deformation and force have been identified. Advantageously, the use of blue and UV light excitation enables adjustment of generated forces that can be maintained even in the dark and can be released by light excitation or temperature. The demonstrated ability to generate light-responsive elements quickly delivering sufficient work paves the way for implementing remotely addressed mechanical functions to future soft robotics and engineering.

Genome-wide identification, characterization, and expression analysis of the expansin gene family in watermelon (Citrullus lanatus).

Expansins are plant cell-wall loosening proteins involved in cell enlargement, adaptive responses to environmental stimuli, and various developmental processes. Although expansins have been characterized in many plant species, little is reported on this family in watermelon. In this study, 30 expansin genes in the watermelon genome (ClEXPs) were identified. These genes which were divided into four subfamilies (7 ClEXLAs, 2 ClEXLBs, 18 ClEXPAs, and 3 ClEXPBs) are unevenly distribute on 10 of 11 watermelon chromosomes. Chromosome mapping suggested that tandem duplication events may have played important roles in the expanding of watermelon expansins. Gene structure and motif identification revealed that same subfamily and subgroup have conserved gene structure and motif. Detection of cis-acting elements revealed that ClEXPs gene promoter regions were enriched with light-responsive elements, hormone-responsive, environmental stress-related, and development-related elements. Expression patterns of ClEXPs were investigated by qRT-PCR. The results showed that expression patterns of 15 ClEXP genes differed in three tissues. Through our own and public RNA-seq analysis, we found that ClEXPs had different expression patterns in fruit flesh, fruit rind, and seed at various developmental stages, and most of ClEXPs were highly responsive to abiotic and biotic stresses. Remarkably, 7 ClEXPs (ClEXLA1, ClEXLA6, ClEXLB1, ClEXLB2, ClEXPA5, ClEXPA10, and ClEXPA16) exhibited positive response to at least three kinds of stresses, suggesting that they might play important roles in the crosstalk of stress signal pathways. The results of this study provide useful insights for the functional identification of expansin gene family in watermelon.

Light Regulation of Two New Manganese Peroxidase-Encoding Genes in Trametes polyzona KU-RNW027.

To better understand the light regulation of ligninolytic systems in Trametes polyzona KU-RNW027, ligninolytic enzymes-encoding genes were identified and analyzed to determine their transcriptional regulatory elements. Elements of light regulation were investigated in submerged culture. Three ligninolytic enzyme-encoding genes, mnp1, mnp2, and lac1, were found. Cloning of the genes encoding MnP1 and MnP2 revealed distinct deduced amino acid sequences with 90% and 86% similarity to MnPs in Lenzites gibbosa, respectively. These were classified as new members of short-type hybrid MnPs in subfamily A.2 class II fungal secretion heme peroxidase. A light responsive element (LRE), composed of a 5′-CCRCCC-3′ motif in both mnp promoters, is reported. Light enhanced MnP activity 1.5 times but not laccase activity. The mnp gene expressions under light condition increased 6.5- and 3.8-fold, respectively. Regulation of laccase gene expression by light was inconsistent with the absence of LREs in their promoter. Blue light did not affect gene expressions but impacted their stability. Reductions of MnP and laccase production under blue light were observed. The details of the molecular mechanisms underlying enzyme production in this white-rot fungus provide useful knowledge for wood degradation relative to illumination condition. These novel observations demonstrate the potential of enhancing ligninolytic enzyme production by this fungus for applications with an eco-friendly approach to bioremediation.

Analysis, Cloning and Prediction of CIS Acting Regulatory Elements of Arabidopsis thaliana Tubulin Gene 4

Tubulin is one of the most abundant member of small family of globular proteins. The most common members of the tubulin family are the proteins that make up microtubules, alpha tubulin and beta tubulin. In this study, we report the identification, analysis and characterization of tubulin geneselected from beta family of Arabidopsis thaliana. Out of various genes, the beta-tubulin gene 4 with 2936bp size was selected. The selected gene was analyzed by using various bioinformatics tools. Moreover, various light responsive cis regulatory elements were found in promoter region of this gene. Many of the genes showed its evolution through gene duplication events.Sequence determination of the Arabidopsis gene revealed a pattern of highly conserved regions. The DNA was isolated from young and fresh leaves of Arabidopsis thaliana plants. Moreover, it was searched in NCBI-Genebank repository for identification and analysis.Further, it was cloned in 5.8 kb expression vector (pGR1) for studying the expression of a reporter gene (GUS). The promoters isolated and cloned by these studies can be used for understanding transcriptional regulation and gene expression studies.

Genome-wide identification and expression analyses of Sm genes reveal their involvement in early somatic embryogenesis in Dimocarpus longan Lour.

The Sm proteins are a conserved protein family with Sm motifs. The family includes Sm and Sm-like proteins, which play important roles in pre-mRNA splicing. Most research on the Sm proteins have been conducted in herbaceous plants, and less in woody plants such as Dimocarpus longan (longan). And the embryo development status significantly affects the quality and yield of longan. In this study, we conducted a genome-wide analysis of longan Sm genes (DlSm) to clarify their roles during somatic embryogenesis (SE) and identified 29 Sm genes. Phylogenetic analysis deduced longan Sm proteins clustered into 17 phylogenetic groups with the homologous Sm proteins of Arabidopsis thaliana. We also analyzed the gene structures, motif compositions, and conserved domains of the longan Sm proteins. The promoter sequences of the DlSm genes contained many light, endosperm development, hormone, and temperature response elements, which suggested their possible functions. In the non-embryogenic callus(NEC) and during early SE in longan, the alternative splicing(AS) events of DlSm genes indicated that these genes may influence SE development by changing gene structures and sequences. The kinetin(KT) hormone, and blue and white light treatments affected the differentiation and growth of longan embryonic callus(EC) probably by affecting the AS events of DlSm genes. Expression profiles showed the possible functional divergence among Sm genes, and different hormones and light qualities affected their expression levels. The expression trends of the DlSm genes determined by RNA sequencing as fragments per kilobase of exon model per million mapped reads (FPKM) and by real-time quantitative PCR(qRT-PCR) during early SE in longan showed that the expression of the DlSm genes was affected by the growth and differentiation of longan SE, and decreased as the somatic embryo differentiation progressed. The results will contributed to understanding the longan Sm gene family and provide a basis for future functional validation studies.

Optical induction of autophagy via Transcription factor EB (TFEB) reduces pathological tau in neurons.

Pathological accumulation of microtubule associated protein tau in neurons is a major neuropathological hallmark of Alzheimer’s disease (AD) and related tauopathies. Several attempts have been made to promote clearance of pathological tau (p-Tau) from neurons. Transcription factor EB (TFEB) has shown to clear p-Tau from neurons via autophagy. However, sustained TFEB activation and autophagy can create burden on cellular bioenergetics and can be deleterious. Here, we modified previously described two-plasmid systems of Light Activated Protein (LAP) from bacterial transcription factor—EL222 and Light Responsive Element (LRE) to encode TFEB. Upon blue-light (465 nm) illumination, the conformation changes in LAP induced LRE-driven expression of TFEB, its nuclear entry, TFEB-mediated expression of autophagy-lysosomal genes and clearance of p-Tau from neuronal cells and AD patient-derived human iPSC-neurons. Turning the blue-light off reversed the expression of TFEB-target genes and attenuated p-Tau clearance. Together, these results suggest that optically regulated TFEB expression unlocks the potential of opto-therapeutics to treat AD and other dementias.

Molecular cloning, functional characterization and transcriptional regulation of the promoter of dihydroflavonal-4-reductase gene from purple-fleshed sweet potato

Dihydroflavonol-4-reductase (DFR) is an important enzyme in the biosynthesis pathway of anthocyanin. It catalyzes the reduction of the dihydroflavonols (dihydrokaempferol, dihydroquercetin and dihydromyricetin) into leucoanthocyanidins, which are precursors for anthocyanins synthesis. In this work, the promoter of gene IbDFR (pIbDFR) was obtained from purple-fleshed sweet potato cv. A5 by high-efficiency thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). Besides the typical promoter elements such as TATA-boxes and CAAT-boxes, there were also many other elements in the sequence of pIbDFR, including one Gibberellin-responsive element, five light-responsive elements, six MYB recognition sites and seven bHLH recognition sites. GUS histochemical assays indicated that the promoter activity was located in the region from position −897 bp to −242 bp of pIbDFR. In order to study the effects of environmental factors on the IbDFR promoter, the transgenic plants of Arabidopsis thaliana were treated with gibberellin, light, sucrose and methyl jasmonate, respectively. The results showed that gibberellin and strong light could promote the activation of pIbDFR. 1 M and 10 M methyl jasmonate (MeJA) inhibited IbDFR promoter activity, while 100 M methyl jasmonate promoted pIbDFR promoter activity. Dual luciferase assay with the pIbDFR was conducted to predict the transcriptional regulating mechanism of transcript factors. The results indicated that the activity of PIbDFR was promoted by IbMYB1 and IbbHLH2.

Molecular Characterization and Promoter Analysis of Novel Sugar Transporter Gene ScERD6 in Sugarcane

Plant sugar transporters are key physiological elements in transporting sucrose and sensing specific sucrose signals. A knockoff of the novel sugar transporter gene (ScERD6) was developed by RACE-PCR from sugarcane, which incorporated an ORF of 1521 bp encoding a protein of 506 amino acids. The isolated ScERD6 gene was measured by 4722 bp DNA and containing 17 exons and 16 introns. It was found that the ScERD6 only had one transcript, and the ScERD6 protein consisted of potentially 12 transmembrane domains, belonging to sugar transporter protein. The promoter sequence was cloned by using genome walking, which included CAAT-boxes and TATA-boxes, as well as specific acting elements such as cis-regulatory elements involved in hormone response, seed development, environmental stresses, ACES, G-box, I-box, L-box, and rbcS-CMA7 light response element, etc. The treatments with 15% PEG and 6 °C stress can induce the expression of ScERD6 in the leaf, root, and stem. But ScERD6 expression in stem was different from that in leaf and root of sugarcane. Real-time PCR analysis uncovered the presence of ScERD6 in roots, stems, leaves, inflorescence, and buds, excessively, and the highest transcript level was observed in inflorescence, and the lowest in roots, while the average was detected in the stem. This finding predicted an important role of this gene in reproductive and floral development.

Blue and red light upregulate α-expansin 1 (EXPA1) in transgenic Brassica rapa and its overexpression promotes leaf and root growth in Arabidopsis

Light quality and the cell-growth-related proteins called expansins (EXPs) are two crucial factors in plant growth and development. However, molecular insights into the light responses of EXPs and the influences of light-dependent EXP expression changes on plant growth and development remain scarce. A preliminary analysis using a cDNA microarray revealed that red, blue, violet, and far-red light emitting diode (LED) light exposure and dark periods differentially regulate the expression of 27 Brassica rapa EXPs (BrEXPs). The results indicated that photosynthetically significant red and blue light induce the expression of BrEXPA1, and these results were further confirmed using a semi-qRT-PCR assay. The identification of several light-responsive cis-regulatory elements in the BrEXP promoter sequences suggested that light signals were perceived through these promoter motifs to regulate the gene expression that eventually controls growth and development. To substantiate our hypothesis that the light-dependent regulation of EXPs modulates growth and development, we developed transgenic reporter lines of B. rapa expressing GUS under the BrEXPA1 promoter. A histochemical analysis showed that BrEXPA1-promoter-driven GUS activity was prominent in the leaves, radicles, lateral roots, and flowers of plants grown under monochromatic red or blue light. The biological functions of BrEXPA1 were further tested by overexpressing sense (OE-S) and antisense BrEXPA1 (OE-AS) transcripts in Arabidopsis. The OE-S lines displayed long radicles, wide leaves, and additional rosette leaves compared to the controls, whereas the OE-AS lines exhibited early bolting. Collectively, our results demonstrated the relationship between light and BrEXPs and also the light-dependent role of BrEXPA1 in growth regulation.

Molecular characterization of teosinte branched1 gene governing branching architecture in cultivated maize and wild relatives

We sequenced the entire tb1 gene in six maize inbreds and its wild relatives (parviglumis, mexicana, perennis and luxurians) to characterize it at molecular level. Hopscotch and Tourist transposable elements were observed in the upstream of tb1 in all maize inbreds, while they were absent in wild relatives. In maize, tb1 consisted of 431-443bp 5'UTR, 1101bp coding sequence and 211-219bp 3'UTR. In promoter region, mutations in the light response element in mexicana (~ 35bp and ~ 55bp upstream of TSS) and perennis (at ~ 35bp upstream of TSS) were found. A 6bp insertion at 420bp downstream of the polyA signal site was present among teosinte accessions, while it was not observed in maize. A codominant marker flanking the 6bp InDel was developed, and it differentiated the teosintes from maize. In Tb1 protein, alanine (12.7-14.6%) was the most abundant amino acid with tryptophan as the rarest (0.5-0.9%). The molecular weight of Tb1 protein was 38757.15g/mol except 'Palomero Toluqueno' and HKI1128. R and TCP motifs in Tb1 protein were highly conserved across maize, teosinte and orthologues, while TCP domain differed for tb1 paralogue. Tb1 possessed important role in light-, auxin-, stress-response and meristem identity maintenance. Presence of molecular signal suggested its localization in mitochondria, nucleus and nucleolus. Parviglumis and mexicana shared closer relationship with maize than perennis and luxurians. A highly conserved 59-60 amino acids long bHLH region was observed across genotypes. Information generated here assumes significance in evolution of tb1 gene and breeding for enhancement of prolificacy in maize.