Transactivation Assay In Yeast Research Articles

TaAP2-10, an AP2/ERF transcription factor, contributes to wheat resistance against stripe rust

The AP2/ERF TF (transcription factor) family is involved in regulating plant responses to various biotic and abiotic stresses. Nevertheless, understanding of the function of AP2/ERF TFs in wheat (Triticum aestivum L.) resistance against the obligate biotrophic stripe rust fungus (Puccinia striiformis f. sp tritici, Pst) remains limited. From a wheat–Pst incompatible interaction cDNA library, the transcript of TaAP2-10 was identified to be significantly induced during Pst infection. TaAP2-10, encodes an AP2 TF with two typical AP2-binding domains. There are three homologues of TaAP2-10 in the wheat genome, located on chromosome 6A, 6B and 6D. TaAP2-10 is localized in the nucleus of wheat protoplasts. A transactivation assay in yeast revealed that TaAP2-10 had transcriptional activation activity that was dependent on its C-terminal region. Quantitative real-time PCR (qRT-PCR) analyses verified that the expression of TaAP2-10 was specifically upregulated by avirulent Pst infection but not by virulent Pst, suggesting its role in wheat resistance to Pst. Furthermore, TaAP2-10 is also induced by abiotic stresses and hormone treatments, particularly under PEG4000 and abscisic acid (ABA) treatments, indicating its potential role in facilitating wheat adaptation to environmental stresses. Silencing TaAP2-10 by barley stripe mosaic virus-induced gene silencing (BSMV-VIGS) significantly reduced wheat resistance against Pst, resulting in a decreased reactive oxygen species (ROS) burst, and promoted Pst growth and development. These findings suggest that TaAP2-10, as a nuclear-localized transcription factor, positively regulates wheat resistance to Pst.

Testing anabolic activity, potency and mechanisms of action of the phyto-derived beta 2 agonist higenamine

Higenamine (Hige), a plant derived alkaloid is classified as β2 agonist by the World Anti-Doping Agency (WADA). However, pharmacologic mechanisms of its performance-enhancing activity have not been investigated so far. Therefore, we investigate the anabolic activity and associated molecular mechanisms of Hige in C2C12 myotubes. In differentiated C2C12 cells dose-dependent effects of Hige on myotube size were analyzed. The mRNA expression of genes involved in hypertrophy was measured. For mechanistic studies, β2-adrenoceptor (ADRB2), androgen receptor (AR), and estrogen receptor (ER) inhibitors and dexamethasone (Dexa) were co-incubated and myotube diameter was evaluated. The interaction of Hige with the AR and ER was investigated. Hige treatment significantly increased myotube diameters and stimulated the mRNA expression of hypertrophy-involved genes. In contrast to the ADRB2 inhibitor (ICI 118551), the ER inhibitor ZK 191703, the AR inhibitor Flutamide (Flu), and treatment with Dexa were able to antagonize the Hige-induced increase of myotube diameter. Hige has antagonistic activity in the AR and ER yeast transactivation assay. Our results demonstrate that Hige induces anabolic effects in C2C12 cells but not via the ADRB2. There are indications for a cross talk between Hige and the AR and ER. Future studies are necessary to investigate the involved molecular mechanisms.

Chemical Stability and Bioactivity of tanshinone I, tanshinone IIA, cryptotanshinone and dihydrotanshinone in in vitro test systems

Danshen Si Wu is a Traditional Chinese Medicine used for menopausal complains. Beside tanshinone IIA (Tan IIA), Danshen also contains tanshinone I (Tan I), cryptotanshinone (CT) and dihydrotanshinone (DT). The aim of this study was to compare the biological activity of these tanshinones and to determine their cytotoxicity and genotoxicity. Purities and stabilities of the substances were analyzed by LC-DAD and LC-MS analyses. DT and CT concentrations decreased rapidly in dimethylsulfoxide and were converted to Tan I and Tan IIA, respectively. In aqueous solution concentration of all tanshinones decreased after 24 h. Tan I and Tan IIA showed dose-dependent bioactivity mediated by ERα and ERβ. No cytotoxic and genotoxic effects for Tan I and Tan IIA were detected. In a yeast transactivation assay Tan I and Tan IIA showed antiandrogenic activity. A significant anabolic activity in C2C12 cells could be detected for Tan I and Tan IIA. In conclusion our data provide evidence that Tan I and Tan IIA are the most relevant bioactive tanshinones in Danshen. Our finding that all tanshinones display a certain instability in aqueous solutions is relevant when discussing their potential therapeutic benefits in humans.

Cloning, characterization and functional analysis of NtMYB306a gene reveals its role in wax alkane biosynthesis of tobacco trichomes and stress tolerance.

Trichomes are specialized hair-like organs found on epidermal cells of many terrestrial plants, which protect plant from excessive transpiration and numerous abiotic and biotic stresses. However, the genetic basis and underlying mechanisms are largely unknown in Nicotiana tabacum (common tobacco), an established model system for genetic engineering and plant breeding. In present study, we identified, cloned and characterized an unknown function transcription factor NtMYB306a from tobacco cultivar K326 trichomes. Results obtained from sequence phylogenetic tree analysis showed that NtMYB306a-encoded protein belonged to S1 subgroup of the plants’ R2R3-MYB transcription factors (TFs). Observation of the green fluorescent signals from NtMYB306a-GFP fusion protein construct exhibited that NtMYB306a was localized in nucleus. In yeast transactivation assays, the transformed yeast containing pGBKT7-NtMYB306a construct was able to grow on SD/-Trp-Ade+X-α-gal selection media, signifying that NtMYB306a exhibits transcriptional activation activity. Results from qRT-PCR, in-situ hybridization and GUS staining of transgenic tobacco plants revealed that NtMYB306a is primarily expressed in tobacco trichomes, especially tall glandular trichomes (TGTs) and short glandular trichomes (SGTs). RNA sequencing (RNA-seq) and qRT-PCR analysis of the NtMYB306a-overexpressing transgenic tobacco line revealed that NtMYB306a activated the expression of a set of key target genes which were associated with wax alkane biosynthesis. Gas Chromatography–Mass Spectrometry (GC-MS) exhibited that the total alkane contents and the contents of n-C28, n-C29, n-C31, and ai-C31 alkanes in leaf exudates of NtMYB306a-OE lines (OE-3, OE-13, and OE-20) were significantly greater when compared to WT. Besides, the promoter region of NtMYB306a contained numerous stress-responsive cis-acting elements, and their differential expression towards salicylic acid and cold stress treatments reflected their roles in signal transduction and cold-stress tolerance. Together, these results suggest that NtMYB306a is necessarily a positive regulator of alkane metabolism in tobacco trichomes that does not affect the number and morphology of tobacco trichomes, and that it can be used as a candidate gene for improving stress resistance and the quality of tobacco.

Overexpression of durum wheat NAC transcription factor TtNTL3A promotes early flowering and increases multiple stress tolerance in transgenic Arabidopsis

Plant-specific NAC transcription factors play important roles in plant development processes, hormone signaling and response to biotic and abiotic stresses. Here, a newly identified membrane-bound NAC gene, designated as TtNTL3A, was isolated from durum wheat. TtNTL3A was homologous to bread wheat TaNAC8 and rice OsNAC8. Moreover, yeast trans-activation assays revealed that TtNTL3A is a transcriptional activator. TtNTL3A was highly expressed in developing seeds and was induced by abiotic stresses, abscisic acid treatment and the infection with Fusarium graminearum. Besides, Transgenic Arabidopsis overexpressing TtNTL3A showed early flowering phenotype and higher tolerance to salt and drought stresses. RT-qPCR analysis revealed that drought and salt-responsive genes were highly expressed in transgenic lines compared to WT plants. Besides, these lines showed resistance to Fusarium graminearum, which was accompanied by a higher expression of pathogenesis-related genes (AtPR-1, Atpdf1.2, and AtNPR1) in TtNTL3A-OE lines. These findings suggest that TtNTL3A is an interesting target of genetic engineering to improve wheat tolerance to biotic and abiotic stresses.

Rice NAC17 transcription factor enhances drought tolerance by modulating lignin accumulation

Land plants have developed a comprehensive system to cope with the drought stress, and it is operated by intricate signaling networks, including transcriptional regulation. Herein, we identified the function of OsNAC17, a member of NAC (NAM, ATAF, and CUC2) transcription factor family, in drought tolerance. OsNAC17 is localized to the nucleus, and its expression was significantly induced under drought conditions. A transactivation assay in yeast revealed that the OsNAC17 is a transcriptional activator, harboring an activation domain in the C-terminal region. Overexpressing (OsNAC17OX) transgenic plants showed drought-tolerant, and knock-out (OsNAC17KO) plants exhibited drought susceptible phenotype compared to non-transgenic plants. Further investigation revealed that OsNAC17 positively regulates several lignin biosynthetic genes and promotes lignin accumulation in leaves and roots. Together, our results show that OsNAC17 contributes to drought tolerance through lignin biosynthesis in rice.

Overexpression of PeNAC122 gene promotes wood formation and tolerance to osmotic stress in poplars

Finding the adequate balance between wood formation and abiotic stress resistance is still an important challenge for industrial woody crops. In this study, PeNAC122, a member of the NAC transcription factor (TF) family highly expressed in xylem, was cloned from Populus euphratica. Tissue expression and β-glucuronidase (GUS) staining showed that PeNAC122 was exclusively expressed in phloem fiber and secondary xylem of stems. Subcellular and yeast transactivation assays confirmed that PeNAC122 protein existed in the nucleus and did not have transcriptional activation and inhibitory activity. Overexpression of PeNAC122 poplar lines exhibited reduced plant height, thickened xylem, and accumulated lignin content in stems, and also upregulates the expression of secondary cell wall biosynthetic genes. Moreover, overexpression of PeNAC122 lines displayed more tolerance to PEG6000-induced osmotic stress, with stronger photosynthetic performance, higher antioxidant enzyme activity, and less accumulation of reactive oxygen species in leaves, and higher expression levels of stress response genes DREB2A, RD29, and NCED3. These results indicate that PeNAC122 plays a crucial role in wood formation and abiotic stress tolerance, which, in addition to potential use in improving wood quality, provides further insight into the role of NAC family TFs in balancing wood development and abiotic stress resistance.

Cloning and Functional Verification of CmRAX2 Gene Associated with Chrysanthemum Lateral Branches Development.

Chrysanthemum (Chrysanthemum morifolium), as one of the four major cut flowers in the world, occupies a large position in the world’s fresh cut flower market. The RAX2 gene is an R2R3 MYB transcription factor that is associated with the development of the axillary bud. In this study, the CmRAX2 gene cloned by homologous cloning in Chrysanthemum morifolium ‘Jinba’ is localized in the nucleus and cytoplasm, having a complete open reading frame (ORF) of 1050 bp and encoding 350 amino acids. The transactivation assay in yeast indicates that CmRAX2 is a transcriptional activator. Quantitative Real-Time PCR (qRT-PCR) Analysis indicated that CmRAX2 was preferentially expressed in the lateral branches and roots of Chrysanthemum morifolium ‘Jinba’, 14.11 and 10.69 times more than in leaves. After the overexpression vector of CmRAX2 was constructed and transformed into Chrysanthemum morifolium ‘Jinba’, it was found that the number of lateral branches and plant height increased, and the emergence time of lateral branches and rooting time advanced after the overexpression of CmRAX2. The results showed that CmRAX2 can promote the lateral bud development of the chrysanthemum, which provides an important theoretical basis for the subsequent molecular breeding and standardized production of the chrysanthemum.

The transcription factor HcERF4 confers salt and drought tolerance in kenaf (Hibiscus cannabinus L.)

Ethylene response factors (ERF) are members of the APETALA2/ERF transcription factor family, and they play an important role in plant growth, development, and response to various environmental stresses. In this study, an ERF transcription factor HcERF4 was isolated and characterized from kenaf. The results showed HcERF4 contained an open reading frame of 702 bp, which comprised of single AP2 domain. Subcellular localization analysis revealed that the HcERF4 gene was localized in the nucleus and cytoplasm. Transactivation assays in yeast demonstrated that HcERF4 functions as a transcriptional activator. Its expression profile was investigated by real-time PCR analysis in different tissues and under salt as well as drought stress condition. The result revealed that HcERF4 could be markedly induced by salt and drought in a time and concentration dependent manner. Moreover, silencing of HcERF4 by using the VIGS technique, caused a significant growth inhibition under salt or drought stress condition, with lower plant height, stem diameter, and fresh weight, indicating HcERF4 plays key roles response abiotic stress. In addition, physiological index analyses showed that the contents of MDA, O2−, H2O2, and free proline significantly increased, while the activities of antioxidant enzymes were significantly restrained in HcERF4 silenced plants. Furthermore, the expression level of eight important genes involved in plant growth regulation, including HcERF3, HcNAC29, HcSOD, HcP5CS, HcbZIP43, HcWRKY31, HcMYB44, and HcCBP25 were investigated, and the result showed that all eight genes were highly expressed in the leaves of the wild kenaf, but were significantly downregulated in the leaves of the VIGS kenaf plants under drought and salt stress conditions; Consequently, the present results indicate HcERF4 plays a positive role in regulating salt and drought stress in kenaf.

Function analysis of ZmNAC33, a positive regulator in drought stress response in Arabidopsis

Drought significantly affects plant growth and has devastating effects on crop production, NAC transcription factors respond to abiotic stresses by activating gene expression. In this study, a maize NAC transcription factor, ZmNAC33, was cloned and characterized its function in Arabidopsis. Transient transformation in Arabidopsis leaves mesophyll protoplasts and trans-activation assays in yeast showed that ZmNAC33 was localized in the nucleus and had transactivation activity. qRT-PCR analysis showed that ZmNAC33 in maize was induced by drought, high salinity and abscisic acid (ABA) stress. Promoter analysis identified multiple stress-related cis-acting elements in the promoter region of ZmNAC33. In ZmNAC33 transgenic Arabidopsis, germination rates were higher than in wild type plants under ABA and osmotic stress at the germination stage, and overexpression lines exhibited higher survival rates and higher antioxidant enzyme activities compared with wild type under drought stress. These results indicate that ZmNAC33 actes as a positive regulator in drought tolerance in plants.

Small Grain and Dwarf 2, encoding an HD-Zip II family transcription factor, regulates plant development by modulating gibberellin biosynthesis in rice

Homeodomain leucine zipper (HD-Zip) proteins are transcription factors that regulate plant development. Bioactive gibberellin (GA) is a key endogenous hormone that participates in plant growth. However, the relationship between HD-Zip genes and modulation of GA biosynthesis in rice remains elusive. Here, we identified a rice mutant, designated as small grain and dwarf 2 (sgd2), which had reduced height and grain size compared with the wild type. Cytological observations indicated that the defective phenotype was mainly due to decreased cell length. Map-based cloning and complementation tests demonstrated that a 9 bp deletion in a homeodomain leucine zipper (HD-Zip) II family transcription factor was responsible for the sgd2 mutant phenotype. Expression of SGD2 was pronounced in developing panicles, and its protein was localized in nucleus. Luciferase reporter system and transactivation assays in yeast suggested that SGD2 functioned as a transcriptional repressor. High performance liquid chromatography assays showed that the endogenous GA1 level in the sgd2 mutant was dramatically decreased, and exogenous GA3 recovered the second leaf sheath to normal length. Results of qRT-PCR showed that the expression levels of genes positively regulating GA-biosynthesis were mostly down-regulated in the mutant. Our data identified the role of an HD-Zip transcription factor that affects rice plant development by modulating gibberellin biosynthesis.

Dissecting the Role of a Basic Helix-Loop-Helix Transcription Factor, SlbHLH22, Under Salt and Drought Stresses in Transgenic Solanum lycopersicum L.

Environmental stresses, such as temperature, heavy metals, drought, cold, and microbial infections adversely damage various aspects of plant growth and development. Salinity and drought are among major hazardous factors, which adversity affects plant growth and productivity. Transcription factors, such as basic helix-loop-helix play critical roles in regulating plant physiological processes under abiotic stresses. In this study, we presented the characterization of a tomato SlbHLH22 gene under abiotic stresses such as drought and salinity. Plants overexpressing SlbHLH22 showed short height with small leaves and enhanced flavonoid accumulation. In wild type (WT) plant, the elevated levels of SlbHLH22 were detected under salt and D-mannitol stresses. Subcellular localization analysis revealed that SlbHLH22 protein was targeted to the nucleus in onion epidermal cells. Transactivation assay in yeast demonstrated that SlbHLH22 had transcriptional activation ability. The transgenic plants overexpressing SlbHLH22 displayed enhanced vigor and more tolerant to drought and salinity than WT. Overexpression of SlbHLH22 significantly peaked the activities of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) to minimize the impacts of reactive oxygen species such as H2O2, which was reduced significantly in transgenic plants along with Malondialdehyde (MDA). Moreover, the expression levels of ROS defense genes (SlPOD, SlCAT, SlSOD), ABA biosynthesis genes, proline biosynthesis, and flavonoids synthesis genes were also activated under salinity and drought. Taken together, our study implies that the overexpression of SlbHLH22 improved tomato plant stress resistance by improving ROS scavenging system, increasing osmotic potential and enhanced accumulation of secondary metabolites in tomato plants.

ScDREB10, an A-5c type of DREB Gene of the Desert Moss Syntrichia caninervis, Confers Osmotic and Salt Tolerances to Arabidopsis.

Drought and salinity are major factors limiting crop productivity worldwide. DREB (dehydration-responsive element-binding) transcription factors play important roles in plant stress response and have been identified in a wide variety of plants. Studies on DREB are focused on the A-1 (DREB1) and A-2 (DREB2) groups. Studies on A-5 group DREBs, which represent a large proportion of the DREB subfamily, is limited. In this study, we characterized and analyzed the stress tolerance function of ScDREB10, an A-5c type DREB gene from the desert moss Syntrichia caninervis. Transactivation assay in yeast showed that ScDREB10 had transactivation activity. Transient expression assay revealed that ScDREB10 was distributed both in the nucleus and cytosol of tobacco leaf epidermal cells. Overexpression of ScDREB10 significantly increased the germination percentage of Arabidopsis seeds under osmotic and salt stresses, and improved the osmotic and salt stress tolerances of Arabidopsis at the seedling stage and is associated with the expression of downstream stress-related genes and improved reactive oxygen species (ROS) scavenging ability. Our study provides insight into the molecular mechanism of stress tolerance of A-5 type DREB proteins, as well as providing a promising candidate gene for crop salt and drought stress breeding.

ZmbZIP4 Contributes to Stress Resistance in Maize by Regulating ABA Synthesis and Root Development.

In plants, bZIP (basic leucine zipper) transcription factors regulate diverse processes such as development and stress responses. However, few of these transcription factors have been functionally characterized in maize (Zea mays). In this study, we characterized the bZIP transcription factor gene ZmbZIP4 from maize. ZmbZIP4 was differentially expressed in various organs of maize and was induced by high salinity, drought, heat, cold, and abscisic acid treatment in seedlings. A transactivation assay in yeast demonstrated that ZmbZIP4 functioned as a transcriptional activator. A genome-wide screen for ZmbZIP4 targets by immunoprecipitation sequencing revealed that ZmbZIP4 could positively regulate a number of stress response genes, such as ZmLEA2, ZmRD20, ZmRD21, ZmRab18, ZmNHX3, ZmGEA6, and ZmERD, and some abscisic acid synthesis-related genes, including NCED, ABA1, AAO3, and LOS5 In addition, ZmbZIP4 targets some root development-related genes, including ZmLRP1, ZmSCR, ZmIAA8, ZmIAA14, ZmARF2, and ZmARF3, and overexpression of ZmbZIP4 resulted in an increased number of lateral roots, longer primary roots, and an improved root system. Increased abscisic acid synthesis by overexpression of ZmbZIP4 also can increase the plant's ability to resist abiotic stress. Thus, ZmbZIP4 is a positive regulator of plant abiotic stress responses and is involved in root development in maize.

A tomato ERF transcription factor, SlERF84, confers enhanced tolerance to drought and salt stress but negatively regulates immunity against Pseudomonas syringae pv. tomato DC3000

ERF proteins are plant-specific transcription factors that play significant roles in plant defense against various stresses. However, only little information regarding stress-related ERF genes is available in tomato (Solanum lycopersicum, Sl). In this study, a tomato ERF gene, SlERF84, was cloned and functionally characterized. The nucleus localization of SlERF84-sGFP was confirmed through a transient expression assay. Transactivation assays in yeast demonstrated that SlERF84 functions as a transcriptional activator. Real-time PCR analysis revealed that SlERF84 could be markedly induced by drought, salt and by several phytohormones (ABA, MeJA and ACC). Overexpression of SlERF84 in Arabidopsis endows transgenic plants with ABA hypersensitivity and enhanced tolerance to drought and salt stress. Histochemical staining assay showed that SlERF84 renders transgenic plants better ROS-scavenging capability. Pathogen inoculation assay revealed that SlERF84 might negatively modulate plant defense response to Pseudomonas syringae pv. tomato DC3000. Moreover, the transcript levels of pathogenesis-related genes AtPR1 and AtPR3 were compromised in transgenic Arabidopsis, as compared to that in Col-0 plants when inoculated with Pseudomonas syringae pv. tomato DC3000. These results suggest that SlERF84 functions as a stress-responsive transcription factor in differentially modulation of abiotic and biotic stress tolerance, and may have applications in the engineering of economically important crops.

Overexpression of the TaSHN1 transcription factor in bread wheat leads to leaf surface modifications, improved drought tolerance, and no yield penalty under controlled growth conditions.

Transcription factors regulate multiple networks, mediating the responses of organisms to stresses, including drought. Here, we investigated the role of the wheat transcription factor TaSHN1 in crop growth and drought tolerance. TaSHN1, isolated from bread wheat, was characterized for molecular interactions and functionality. The overexpression of TaSHN1 in wheat was followed by the evaluation of T2 and T3 transgenic lines for drought tolerance, growth, and yield components. Leaf surface changes were analysed by light microscopy, SEM, TEM, and GC-MS/GC-FID. TaSHN1 behaves as a transcriptional activator in a yeast transactivation assay and binds stress-related DNA cis-elements, determinants of which were revealed using 3D molecular modelling. The overexpression of TaSHN1 in transgenic wheat did not result in a yield penalty under the controlled plant growth conditions of a glasshouse. Transgenic lines had significantly lower stomatal density and leaf water loss and exhibited improved recovery after severe drought, compared with control plants. The comparative analysis of cuticular waxes revealed an increased accumulation of alkanes in leaves of transgenic lines. Our data demonstrate that TaSHN1 may operate as a positive modulator of drought stress tolerance. Positive attributes could be mediated through an enhanced accumulation of alkanes and reduced stomatal density.

Characterization and functional analysis of GhWRKY42, a group IId WRKY gene, in upland cotton (Gossypium hirsutum L.)

BackgroundWRKY transcription factors (TFs) participate in various physiological processes of plants. Although WRKY genes have been well studied in model plants, knowledge of the functional roles of these genes is still extremely limited in cotton.ResultsIn this study, a group IId WRKY gene from cotton, GhWRKY42, was isolated and characterized. Our data showed that GhWRKY42 localized to the nucleus. A transactivation assay in yeast demonstrated that GhWRKY42 was not a transcriptional activator. A β-glucuronidase (GUS) activity assay revealed that the promoter of GhWRKY42 showed fragment deletion activity in Nicotiana tabacum and was mainly expressed in the roots, stems and leaves of ProGhWRKY42::GUS transgenic Arabidopsis plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that GhWRKY42 was up-regulated during leaf senescence and was induced after exposure to abiotic stresses. Constitutive expression of GhWRKY42 in Arabidopsis led to a premature aging phenotype, which was correlated with an increased number of senescent leaves, reduced chlorophyll content and elevated expression of senescence-associated genes (SAGs). In addition, virus-induced gene silencing (VIGS) was used to silence the endogenous GhWRKY42 gene in cotton, and this silencing reduced plant height.ConclusionsOur findings indicate that GhWRKY42 is involved in abiotic stress responses, premature leaf senescence and stem development. This work establishes a solid foundation for further functional analysis of the GhWRKY42 gene in cotton.

Functional analysis of the HD-Zip transcription factor genes Oshox12 and Oshox14 in rice.

The homeodomain-leucine zipper (HD-Zip) transcription factor family plays vital roles in plant development and morphogenesis as well as responses to biotic and abiotic stresses. In barley, a recessive mutation in Vrs1 (HvHox1) changes two-rowed barley to six-rowed barley, which improves yield considerably. The Vrs1 gene encodes an HD-Zip subfamily I transcription factor. Phylogenetic analysis has shown that the rice HD-Zip I genes Oshox12 and Oshox14 are the closest homologues of Vrs1. Here, we show that Oshox12 and Oshox14 are ubiquitously expressed with higher levels in developing panicles. Trans-activation assays in yeast and rice protoplasts demonstrated that Oshox12 and Oshox14 can bind to a specific DNA sequence, AH1 (CAAT(A/T)ATTG), and activate reporter gene expression. Overexpression of Oshox12 and Oshox14 in rice resulted in reduced panicle length and a dwarf phenotype. In addition, Oshox14 overexpression lines showed a deficiency in panicle exsertion. Our findings suggest that Oshox12 and Oshox14 may be involved in the regulation of panicle development. This study provides a significant advancement in understanding the functions of HD-Zip transcription factors in rice.

Overexpression of a SBP-Box Gene (VpSBP16) from Chinese Wild Vitis Species in Arabidopsis Improves Salinity and Drought Stress Tolerance.

Salinity and drought are two major abiotic stresses that limit grape productivity. Responses to stress in grape are known to be regulated by several families of transcription factors. However, little is known about the role of grape Squamosa promoter binding protein (SBP)-box transcription factor genes in response to abiotic stress. To better understand the functions of the grape SBP-box genes in abiotic stress tolerance, a full-length complementary DNA (cDNA) sequence of the putative SBP-box transcription factor gene, VpSBP16 was amplified from Chinese wild grapevine Vitis pseudoreticulata clone “Baihe-35-1”. We observed that the VpSBP16 protein fused to the green fluorescent protein (GFP) reporter accumulated in the nucleus when transiently expressed in onion epidermal cells. Moreover, VpSBP16 was shown to have transcriptional activation activity using a yeast trans-activation assay. We performed a VpSBP16 functional analysis through the characterization of transgenic Arabidopsis thaliana plants constitutively over-expressing VpSBP16. The transgenic lines had longer roots and the seeds had a higher germination rate than the wild type (WT) under osmotic stress. In addition, the accumulation of reactive oxygen species (ROS) of transgenic seedlings was significantly lower than WT in the transgenic lines, as was electrolyte leakage. VpSBP16 overexpression also elevated expression levels of stress-response genes involved in the salt overly sensitive (SOS) pathway. These results indicate that overexpression VpSBP16 in A. thaliana enhances tolerance of salt and drought stress during seed germination, as well in seedlings and mature plants, by regulating SOS and ROS signaling cascades.

A bHLH Transcription Factor Regulates Bisbibenzyl Biosynthesis in the Liverwort Plagiochasma appendiculatum.

Liverworts, a section of the bryophyte plants which pioneered the colonization of terrestrial habitats, produce cyclic bisbibenzyls as secondary metabolites. These compounds are generated via the phenylpropanoid pathway, similar to flavonoid biosynthesis, for which basic helix-loop-helix (bHLH) transcription factors have been identified as one of the important regulators in higher plants. Here, a bHLH gene homolog (PabHLH) was isolated from the liverwort species Plagiochasma appendiculatum and its contribution to bisbibenzyl biosynthesis was explored. Variation in the abundance of PabHLH transcript mirrored that of tissue bisbibenzyl content in three different liverwort tissues. A phylogenetic analysis based on the bHLH domain sequence suggested that the gene encodes a member of bHLH subgroup IIIf, which clusters proteins involved in flavonoid synthesis. The gene's transient expression in onion epidermal cells implied that its product localized to the nucleus, and a transactivation assays in yeast showed that it was able to activate transcription. In both callus and thallus, the overexpression of PabHLH boosted bisbibenzyl accumulation, while also up-regulating PaPAL, Pa4CL1, PaSTCS1 and two genes encoding P450 cytochromes, and its RNA interference (RNAi)-induced suppression down-regulated the same set of genes and reduced the accumulation of bisbibenzyls. The abundance of PaCHS and PaFNSI transcript was related to flavonoid accumulation in transgenic thallus. PabHLH represents a candidate for the metabolic engineering of bisbibenzyl content.