GUS Expression Research Articles

Overexpression of MusaNAC68 reduces secondary wall thickness of xylem tissue in banana

Secondary wall is an important component of xylem tissue, as it maintains the integrity of the vessel elements and provides mechanical strength for upward growth of plants. Enough prior evidence has linked secondary wall thickening with auxin-signaling pathway. Hence, plants showing auxin-mediated effects might demonstrate alteration of secondary wall depositions. GUS expression from promoter of NAC68 (pMusaNAC68) was observed in many organs with strong expression in vascular tissues. Transgenic banana plants overexpressing NAC68 transcription factor were analyzed for lignin and secondary wall depositions. Strong reduction of lignin in cross-sections of transgenic lines was observed by phloroglucinol-HCl and toluidine blue-O staining. The reduction of secondary wall thickness observed after calcofluor white staining was substantiated by data from scanning electron microscopy indicating reproducibly lower secondary wall depositions. A strong reduction in transcript levels of PAL, 4CL, C4H, COMT and CcOAMT was observed due to the overexpression of NAC68. Furthermore, substantial elevation of MYB transcription factors such as MYB4a-like and MYB4b-like which are probable repressors of phenylpropanoid biosynthesis pathway was also observed. Expression of MYB transcription factors, MYB85-like and MYB58-like which are probable activators of secondary wall depositions, was remarkably downregulated after NAC68 overexpression. Transgenic lines also showed altered expression of genes coding for cellulose synthase subunits with remarkable elevation in expression of a close homologue of Arabidopsis CesA8, which functions in cellulose deposition during secondary wall development. The study indicated a novel function of NAC68 transcription factor in regulating secondary wall thickening and will enhance our knowledge about crosstalk between auxin-signaling and secondary wall depositions.

Agroinfiltration is a key factor to improve the efficiency of apple and pear transformation

Genetic transformation of apple and pear is mainly performed via Agrobacterium tumefaciens. The average efficiency of transformation remains low in most laboratories and the success of the transformation experiments is very variable. Improving the efficiency and the reproducibility of apple and pear transformation is thus highly desirable. Adventitious regeneration ability of the explants is not a limiting factor for apple or pear transformation. The present study focuses on improving the frequency of stably transformed cells in the explants following A. tumefaciens inoculation. We report here the results of 36 independent transformation experiments on ‘Gala’ apple, performed with 10 different binary plasmids, comparing three methods of A. tumefaciens inoculation. Agroinfiltration (Agrobacterium-mediated vacuum infiltration) of the explants in a bacterial suspension containing a surfactant (Silwet L-77) at a low concentration (0.002% v/v) significantly increased the average transformation efficiency (mean rate of 5.8% and maximum rate of 30%) and reduced the number of failed experiments (16%) compared to the method of crushing the explants with non-traumatic forceps prior to immersion into the inoculum. The success of the agroinfiltration method was associated with a very high level of GUS expression one month after inoculation.In addition, agroinfiltration dramatically increased the transformation efficiency of pear, reaching rates of transformation between 50 and 80%, compared to inoculation with a scalpel dipped into the inoculum. Altogether, our results demonstrate that the production of large number of transgenic apple or pear lines in a short period of time is feasible using the agroinfiltration method.

RNA-Seq Transcriptome Analysis of Rice Primary Roots Reveals the Role of Flavonoids in Regulating the Rice Primary Root Growth.

Flavonoids play important roles in root development and in its tropic responses, whereas the flavonoids-mediated changes of the global transcription levels during root growth remain unclear. Here, the global transcription changes in quercetin-treated rice primary roots were analyzed. Quercetin treatment significantly induced the inhibition of root growth and the reduction of H2O2 and O2− levels. In addition, the RNA-seq analysis revealed that there are 1243 differentially expressed genes (DEGs) identified in quercetin-treated roots, including 1032 up-regulated and 211 down-regulated genes. A gene ontology (GO) enrichment analysis showed that the enriched GO terms are mainly associated with the cell wall organization, response to oxidative stress, and response to hormone stimulus. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis showed that the enriched DEGs are involved in phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction. Moreover, the quercetin treatment led to an increase of the antioxidant enzyme activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in rice roots. Also, the quercetin treatment altered the DR5:GUS expression pattern in the root tips. All of these data indicated that the flavonoids-mediated transcription changes of genes are related to the genes involved in cell wall remodeling, redox homeostasis, and auxin signaling, leading to a reduced cell division in the meristem zone and cell elongation in the elongation zone of roots.

WRINKLED1 transcription factor orchestrates the regulation of carbon partitioning for C18:1 (oleic acid) accumulation in Siberian apricot kernel

WRINKLED1 (WRI1), an APETALA2 (AP2)-type transcription factor, has been shown to be required for the regulation of carbon partitioning into fatty acid (FA) synthesis in plant seeds. To our knowledge, the regulatory network of WRI1 remains unknown in Prunus sibirica kernel (PSK), a novel woody biodiesel feedstock in China. In this study, based on the transcriptional data from developing oilseeds of multiple plant species, we identified 161 WRI1-coexpressed genes using weighted gene co-expression network analysis (WGCNA). The major portion of WRI1-coexpressed genes was characterized to be involved in carbon partitioning and FA biosynthesis. Additionally, we detected the temporal patterns for oil content and FA compositions in developing PSK from two different germplasms (AS-85 and AS-86). The major differences between the two germplasms are higher contents of oil and C18:1 in AS-85 than in AS-86 at a mature stage. Thus, AS-85 and AS-86 are desirable materials to explore the molecular and metabolic mechanisms of oil accumulation in Siberian apricot. Expression analysis in developing PSK of AS-85 and AS-86 indicated that the expression level of P. sibirica WRI1 (PsWRI1) was closely correlated to accumulative rate of oil. Also, the comparison of expression profiles in developing PSK of AS-85 and AS-86 displayed that the pPK, E1-α, E2, TAL, BC, MCMT, BS, SAD and FAD2 have a high correlation with PsWRI1. Transient expression showed that ProSAD- and ProBS-driving GUS expression showed no substantial difference between AS-85 and AS-86, while the expression level of ProPEPCK-AS-85 driving GUS was significantly higher than that of ProPEPCK-AS-86 driving GUS. Additionally, transient co-transformation with PsWRI1 revealed that ProSAD, ProPEPCK and ProBS activity could be specifically up-regulated by PsWRI1. This regulatory mechanism of PsWRI1 may create a steep concentration difference, thereby facilitating carbon flux into C18:1 accumulation in developing PSK. Overall, all our findings imply a versatile mechanism of WRI1 to optimize carbon allocation for oil accumulation, which can provide reference for researching the woody biodiesel plants.

Sicwinv1, a Cell Wall Invertase from Sesame, Is Involved in Anther Development

Male sterility is an effective phenotype for sesame hybrid production. Cell wall invertase (CWINV) plays a crucial role in providing carbohydrates for male gametophyte development, and anther-specific repression of CWINV genes in different species causes male sterile plants. In this study, a sesame CWINV gene defined as Sicwinv1 was cloned. Sequence analysis indicated that it contained the conserved β-fructosidase motif and a cysteine catalytic site as well as the glycosylation motif of CWINV proteins. Subcellular localization indicated that Sicwinv1 was localized in the cell wall. Transient overexpression of Sicwinv1 in N. benthamiana plants dramatically increased Sicwinv1 activity in the cell wall of Sicwinv1 overexpression leaves. RT-PCR revealed that Sicwinv1 was particularly expressed in the stamen. Further promoter analysis in Arabidopsis confirmed the anther-specific expression pattern and showed that GUS staining was mainly detected in the anther tapetum. The quantitative GUS assay indicated that the Sicwinv1 promoter drove GUS expression and was correlated with the anther development stage. Consistently, in situ hybridization revealed that Sicwinv1 was expressed in the tetrad stage and microspore stage in sesame and that Sicwinv1 RNA mainly accumulated in the tapetum, and tetrad, microspore, indicating a role of Sicwinv1 in anther development and male fertility. Our results might be an important first step toward unravelling the roles of sesame CWINV and point to a potential for utilizing this gene and its promoter for engineering male sterility for hybrid production in sesame.

Isolation and functional validation of the CmLOX08 promoter associated with signalling molecule and abiotic stress responses in oriental melon, Cucumis melo var. makuwa Makino

BackgroundLipoxygenases (LOXs) play significant roles in abiotic stress responses, and identification of LOX gene promoter function can make an important contribution to elucidating resistance mechanisms. Here, we cloned the CmLOX08 promoter of melon (Cucumis melo) and identified the main promoter regions regulating transcription in response to signalling molecules and abiotic stresses.ResultsThe 2054-bp promoter region of CmLOX08 from melon leaves was cloned, and bioinformatic analysis revealed that it harbours numerous cis-regulatory elements associated with signalling molecules and abiotic stress. Five 5′-deletion fragments obtained from the CmLOX08 promoter—2054 (LP1), 1639 (LP2), 1284 (LP3), 1047 (LP4), and 418 bp (LP5)—were fused with a GUS reporter gene and used for tobacco transient assays. Deletion analysis revealed that in response to abscisic acid, salicylic acid, and hydrogen peroxide, the GUS activity of LP1 was significantly higher than that of the mock-treated control and LP2, indicating that the − 2054- to − 1639-bp region positively regulates expression induced by these signalling molecules. However, no deletion fragment GUS activity was induced by methyl jasmonate. In response to salt, drought, and wounding treatments, LP1, LP2, and LP4 promoted significantly higher GUS expression compared with the control. Among all deletion fragments, LP4 showed the highest GUS expression, indicating that − 1047 to − 1 bp is the major region regulating promoter activity and that the − 1047 to − 418-bp region positively regulates expression induced by salt, drought, and wounding, whereas the − 1284 to − 1047-bp region is a negative regulatory segment. Interestingly, although the GUS activity of LP1 and LP2 was not affected by temperature changes, that of LP3 was significantly induced by heat, indicating that the − 1284- to − 1-bp region is a core sequence responding to heat and the − 2054- to − 1284-bp region negatively regulates expression induced by heat. Similarly, the − 1047- to − 1-bp region is the main sequence responding to cold, whereas the − 2054- to − 1047-bp region negatively regulates expression induced by cold.ConclusionsWe cloned the CmLOX08 promoter and demonstrated that it is a signalling molecule/stress-inducible promoter. Furthermore, we identified core and positive/negative regulatory regions responding to three signalling molecules and five abiotic stresses.

Transgenic Wucai (Brassica campestris L.) produced via Agrobacterium-mediated anther transformation in planta.

We developed a novel Agrobacterium-mediated anther transformation for Wucai in planta, and in this procedure, the male germ line was the predominant target. Wucai (Brassica campestris L.), a variant of non-heading Chinese cabbage, is widely cultured in China and only improved by classic breeding methods. Here, a novel and efficient in planta Agrobacterium-mediated anther transformation method is developed based on the optimization of several factors that affect anther transformation. After optimization, transformation with the manual pollination application led to increased transient GUS expression in anthers (reaching 91.59%) and the transformation efficacies in planta (0.59-1.56% for four commercial cultivars). The stable integration and inheritance of the transgenes were further examined by molecular and genetic analyses. Three T2 transgenic lines presented a segregation ratio of 3:1, which was consistent with the Mendelian feature of a single dominant gene. In addition, the GUS histochemical assay and genetic crossing analysis revealed that the male germ line was the predominant target in this transformation. This optimized transformation system could provide a useful tool for both the improvement of cultivar qualities and investigation of functional genes in Wucai.

Transient transformation of sweet cherry pollen with a hairpin gene construct targetingS haplotype-specific F-box, the pollenSdeterminant ofPrunusself-incompatibility

Sweet cherry (Prunus avium) exhibits gametophytic self-incompatibility (GSI), which inhibits fertilization from self-pollen and pollen containing an S-allele that matches that in the maternal parent. Although several sweet cherry mutants with pollen-part self-compatibility (SC) exist, it is still difficult to break through self-incompatibility (SI), artificially. The pollen determinant gene, S haplotype-specific F-box (SFB), is suggested to be indispensable for self-pollen rejection in Prunus GSI. In the present study, we attempted to confer SC on sweet cherry pollen by silencing PavSFB. A hairpin gene sequence, hpPav6, was designed from a 320-bp fragment of PavSFB-S(6) and Pdk intron. Ectopic expression of hpPav6 reduced the transcript level of PavSFB-S(6) to half in the agroinfiltrated tobacco leaves, which indicated its effectiveness to silence PavSFB-S(6). A plasmid containing LAT52 promoter-driven hpPav6 and beta-glucuronidase (GUS) was constructed to transform pollen using particle bombardment. Particle bombardment of the constructed plasmid induced GUS expression in ∼1% 'Satonishiki' (S(3)S(6)) pollen using an optimized parameter-set. A small particle size (0.6 μm) and high bombarding pressure (1550 psi) tended to improve the transformation efficiency. The bombarded pollen was used for self-pollination; however, clear evidence indicating transformation to SC was not obtained. The tubes of the bombarded pollen reached near the base of the self-style in two out of the 93 tested pistils; however, no fruit set was obtained in over 500 flowers pollinated on the plant.

Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos.

Wheat is the most widely grown staple food crop in the world and accounts fordietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2days, 30min and 48h, respectively. The bacterial inoculum concentration of OD of1at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4mg/l along with antibiotics (cefotaxime 500mg/l and timentin 300mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

LBD16 and LBD18 acting downstream of ARF7 and ARF19 are involved in adventitious root formation in Arabidopsis

BackgroundAdventitious root (AR) formation is a complex genetic trait, which is controlled by various endogenous and environmental cues. Auxin is known to play a central role in AR formation; however, the mechanisms underlying this role are not well understood.ResultsIn this study, we showed that a previously identified auxin signaling module, AUXIN RESPONSE FACTOR(ARF)7/ARF19-LATERAL ORGAN BOUNDARIES DOMAIN(LBD)16/LBD18 via AUXIN1(AUX1)/LIKE-AUXIN3 (LAX3) auxin influx carriers, which plays important roles in lateral root formation, is involved in AR formation in Arabidopsis. In aux1, lax3, arf7, arf19, lbd16 and lbd18 single mutants, we observed reduced numbers of ARs than in the wild type. Double and triple mutants exhibited an additional decrease in AR numbers compared with the corresponding single or double mutants, respectively, and the aux1 lax3 lbd16 lbd18 quadruple mutant was devoid of ARs. Expression of LBD16 or LBD18 under their own promoters in lbd16 or lbd18 mutants rescued the reduced number of ARs to wild-type levels. LBD16 or LBD18 fused to a dominant SRDX repressor suppressed promoter activity of the cell cycle gene, Cyclin-Dependent Kinase(CDK)A1;1, to some extent. Expression of LBD16 or LBD18 was significantly reduced in arf7 and arf19 mutants during AR formation in a light-dependent manner, but not in arf6 and arf8. GUS expression analysis of promoter-GUS reporter transgenic lines revealed overlapping expression patterns for LBD16, LBD18, ARF7, ARF19 and LAX3 in AR primordia.ConclusionThese results suggest that the ARF7/ARF19-LBD16/LBD18 transcriptional module via the AUX1/LAX3 auxin influx carriers plays an important role in AR formation in Arabidopsis.

Molecular identification of a root apical cell-specific and stress-responsive enhancer from an Arabidopsis enhancer trap line

BackgroundPlant root apex is the major part to direct the root growth and development by responding to various signals/cues from internal and soil environments. To study and understand root system biology particularly at a molecular and cellular level, an Arabidopsis T-DNA insertional enhancer trap line J3411 expressing reporters (GFP) only in the root tip was adopted in this study to isolate a DNA fragment.ResultsUsing nested PCR, DNA sequencing and sequence homology search, the T-DNA insertion site(s) and its flanking genes were characterised in J3411 line. Subsequently, a 2000 bp plant DNA-fragment (Ertip1) upstream of the insert position of the coding T-DNA was in silico analysed, revealing certain putative promoter/enhancer cis-regulatory elements. Cloning and transformation of this DNA fragment and its truncated segments tagged with or without 35S minimal promoter (35Smini), all of which were fused with a GFP or GUS reporter, allowed to detect GFP and GUS expression mediated only by Ertip1 + 35mini (PErtip1+35Smini) specifically in the Arabidopsis root tip region. The PErtip1+35Smini activity was further tested to be strong and stable under many different growth conditions but suppressed by cold, salt, alkaline pH and higher ammonium and phosphorus.ConclusionThis work describes a promising strategy to isolate a tissue-/cell-specific enhancer sequence from the enhancer trap lines, which are publically available. The reported synthetic promoter i.e. PErtip1+35Smini may provide a valuable and potent molecular-tool for comprehensive investigation of a gene function related to root growth and development as well as molecular engineering of root-architectural formation aiming to improve plant growth.

Effective Agrobacterium-mediated transformation protocols for callus and roots of halophyte ice plant (Mesembryanthemum crystallinum)

BackgroundIce plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols.ResultsTo establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL−1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33–100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks.ConclusionsOur Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4–3.0% with callus and 33.3–100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.

ZmPBF and ZmGAMYB transcription factors independently transactivate the promoter of the maize (Zea mays) β-carotene hydroxylase 2 gene.

The maize (Zea mays) enzyme β-carotene hydroxylase 2 (ZmBCH2) controls key steps in the conversion of β-carotene to zeaxanthin in the endosperm. The ZmBCH2 gene has an endosperm-preferred and developmentally regulated expression profile, but the detailed regulatory mechanism is unknown. To gain insight into the regulation of ZmBCH2, we isolated 2036bp of the 5'-flanking region containing the 263bp 5'-untranslated region (5'-UTR) including the first intron. We linked this to the β-glucuronidase reporter gene gusA. We found that high-level expression of gusA in rice seeds requires the 5'-UTR for enhanced activation. Truncated variants of the ZmBCH2 promoter retained their seed-preferred expression profile as long as a prolamin box and AACA motif were present. We identified candidate genes encoding the corresponding transcription factors (ZmPBF and ZmGAMYB) and confirmed that their spatiotemporal expression profiles are similar to ZmBCH2. Both ZmPBF and ZmGAMYB can transactivate ZmBCH2 expression in maize endosperm. To eliminate potential confounding effects in maize, we characterized the regulation of the minimal promoter region of ZmBCH2 in transgenic rice. This revealed that ZmPBF and ZmGAMYB independently transactivate the ZmBCH2 promoter. The mechanism that underpins our data provides an exciting new strategy for the control of target gene expression in engineered plants.

The promoter of tomato HISTIDINE DECARBOXYLASE A is fruit-specific, and its expression is stably maintained in fruits during ripening

Identifying novel promoters with specific temporal and spatial expression patterns is crucial for crop biotechnology. In this study, we isolated a fruit-specific promoter in tomato, HISTIDINE DECARBOXYLASE A (SlHDC-A) promoter. Through RNA-seq and RT-PCR analysis, we found that SlHDC-A was predominantly expressed in fruits and that its expression was stable in fruits during ripening. These results suggest that the promoter of SlHDC-A might have the ability to determine fruit-specific gene expression. To test this possibility, we generated transgenic tomato transformed with SlHDC-A::GUS and 35S::GUS. Unlike 35S::GUS transgenic tomato with constitutive expression in various tissues, SlHDC-A::GUS transgenic plants showed fruit-specific expression of GUS. The intensity of GUS activity in fruits of SlHDC-A::GUS transgenic plants was approximately tenfold higher than that in fruits of 35S::GUS transgenic plants. The core region responsible for its fruit-specific expression was identified by promoter deletion analyses. Removal of the − 880 to − 577 region abolished the fruit-specific expression of SlHDC-A promoter. This suggests that the − 880 to − 577 region is the core region responsible for the fruit-specific expression of SlHDC-A. This finding was further supported by analysis of chimeric fusion promoter. Unlike 35S minimal promoter which had no activity to express GUS, the chimeric fusion promoter of the core region and 35S minimal promoter showed fruit-specific expression similar to intact SlHDC-A promoter. Collectively, these findings indicate that the promoter of SlHDC-A is fruit-specific and the − 880 to − 577 region is the core region of SlHDC-A promoter.

Exogenous intervention with sodium hydrosulfite enhances the quality of Radix Scutellariae by modulating antioxidases and secondary metabolites

Introduction: The transformation of wild herbal medicine into cultivation results in decreased quality, becoming an issue of concern in China, even has a word, “traditional Chinese medical science would be destroyed by herbal medicine.” Now, there is no way to acquire superior herb medicine. Reactive oxygen species (ROS) are generated under stress and represent key plant products that induce secondary metabolism. ROS may be important intermediates that link environmental stress to secondary metabolism. Sodium hydrosulfite (Na2S2O4) is just one compound that produces O2•-. Materials and Methods: The fresh roots of Scutellaria baicalensis were treated with 0.0004, 0.04, and 4 mmol/L Na2S2O4, respectively. A stress model was established to elucidate the change of secondary metabolism, antioxidant enzyme system, and enzymes relating to flavonoids. Results: Na2S2O4regulated the activities of β-glucuronidase (GUS) and UDP-glucuronate baicalein 7-O-glucuronosyltransferase (UBGAT) and promoted the transformation of flavonoid glycosides into aglycones with greater antioxidant capacities. In fresh S. baicalensis roots, Na2S2O4decreased the activities of superoxide dismutase, catalase, and peroxidase (POD), which led to a substantial accumulation of hydrogen peroxide (H2O2). H2O2increased the expression of phenylalanine ammonia lyase, GUS, and UBGAT genes and promoted the synthesis and transformation of flavonoids. Moreover, polyphenol oxidase regulated the flavonoid content to maintain the oxidation balance. Although the content of flavonoid glycosides was slightly reduced, the total flavonoid content remained high. Notably, the content of baicalein, an antioxidant compound, increased from 0.28% to 1.96%. Conclusions: ROS facilitated the dominant role of secondary metabolites and enhanced the quality of Radix Scutellariae. Abbreviations used: APX: Ascorbate peroxidase, CAT: Catalase, GUS: β-glucuronidase, PAL: Phenylalanine ammonia lyase, POD: Peroxidase, PPO: Polyphenol oxidase, ROS: Reactive oxygen species, SOD: Superoxide dismutase, UBGAT: UDP-glucuronate baicalein 7-O-glucuronosyltransferase.

Phosphatidylinositol-specific phospholipase C2 functions in auxin-modulated root development.

Nine phosphatidylinositol-specific phospholipases C (PLCs) have been identified in the Arabidopsis genome; among the importance of PLC2 in reproductive development is significant. However, the role of PLC2 in vegetative development such as in root growth is elusive. Here, we report that plc2 mutants displayed multiple auxin-defective phenotypes in root development, including short primary root, impaired root gravitropism, and inhibited root hair growth. The DR5:GUS expression and the endogenous indole-3-acetic acid (IAA) content, as well as the responses of a set of auxin-related genes to exogenous IAA treatment, were all decreased in plc2 seedlings, suggesting the influence of PLC2 on auxin accumulation and signalling. The root elongation of plc2 mutants was less sensitive to the high concentration of exogenous auxins, and the application of 1-naphthaleneacetic acid or the auxin transport inhibitor N-1-naphthylphthalamic acid could rescue the root hair growth of plc2 mutants. In addition, the PIN2 polarity and cycling in plc2 root epidermis cells were altered. These results demonstrate a critical role of PLC2 in auxin-mediated root development in Arabidopsis, in which PLC2 influences the polar distribution of PIN2.

Evaluation of the Genotype-dependency of the Leafbased Regeneration and Transformation System in Maize

Tissue culture and genetic transformation in maize are very laborious. The existing regeneration methods, which mainly use immature embryos as starting material, are highly genotype-dependent. Leaf segments can be used as an alternative explant source to produce embryogenic calli. Although a reliable leafbased regeneration and transformation system has been recently reported for maize, however, the genotype-dependency of this method has not been described yet. To this end, we evaluated the production of embryogenic calli from young leaves of several maize genotypes. The results showed that, overall callus induction potential as well as embryogenic callus induction rate is significantly different among the tested genotypes, demonstrating the genotypedependency of this system. However, induced embryogenic calli from different genotypes remained their embryogenic capability during several callus multiplication rounds. In addition, embryogenic calli showed high potential for biolistic-based genetic transformation, as revealed by transient GUS expression.
 Plant Tissue Cult. & Biotech. 28(2): 261-268, 2018 (December)

Regeneration and Agrobacterium-mediated transformation of japonica rice varieties developed for a cold region

So far, a large number of transformation systems have been established for japonica rice, but only a few have been reported for cold-region varieties. In our study, we established highly efficient tissue culture systems for two cold-region rice cultivars, Dongnong 427 and Longdao 14. Plant growth regulator (PGR) levels were optimized by an orthogonal experimental design. The culture ability, constituted by induction and differentiation rate, served as the detection index of orthogonal experiments. The optimal combinations of PGRs for callus induction and regeneration of Dongnong 427 and Longdao 14 were 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) + 2 mg/l 6-benzyladenine (BA) + 4 mg/l kinetin (KIN) + 0.2 mg/l α-naphthaleneacetic acid (NAA) and 1 mg/l 2,4-D + 4 mg/l 6-BA + 4 mg/l KIN + 0.5 mg/l NAA, respectively. Agrobacterium strain EHA 105 containing the plasmid pCAMBIA1301 was used for transformation. The frequency of transient transformation was expressed as the ratio between the number of calli showing GUS expression and the total number of calli kept for staining. The highest transformation efficiency in Dongnong 427 was obtained when calli were immersed in 0.272 OD<sub>600</sub> (optical density determined at 600 nm) for 10 min. While it was best for Longdao 14 calli to be infected with 0.592 OD<sub>600</sub> for 20 min. Infected calli of the two varieties were co-cultivated on two pieces of sterile filter paper moistened with 1 ml liquid co-cultivation medium for three days. The expression of the GUS gene was confirmed by PCR analysis of plants of both varieties.

The bacterial volatile dimethyl-hexa-decylamine reveals an antagonistic interaction between jasmonic acid and cytokinin in controlling primary root growth of Arabidopsis seedlings.

Chemical communication underlies major adaptive traits in plants and shapes the root microbiome. An increasing number of diffusible and/or volatile organic compounds released by bacteria have been identified, which play phytostimulant or protective functions, including dimethyl-hexa-decylamine (DMHDA), a volatile biosynthesized by Arthrobacter agilis UMCV2 that induces jasmonic acid (JA) signaling in Arabidopsis. Here, he found that the growth repressing effects of both DMHDA and JA are antagonized by kinetin and correlated with an inhibition of cytokinin-related ARR5::GUS and TCS::GFP expression in Arabidopsis primary roots. Moreover, we demonstrate that shoot supplementation of JA triggers JAZ1 expression both locally and systemically and represses cytokinin-dependent promoter activity in roots. A similar effect was observed after cotyledon wounding, in which an increase of JA-inducible LOX2:GUS expression represses root growth, which correlates with the loss of TCS::GFP detection at the very root tip. Our data demonstrate that the bacterial volatile DMHDA crosstalks with cytokinin signaling and reveals the downstream antagonistic interaction between JA and cytokinin in controlling root growth.

Transgenic Lilium longiflorum plants containing the bar-uidA gene controlled by the rice RPC1, Agrobacterium rolD, mas2, and CaMV 35S promoters

Lilies are grown in the field for bulb production where they are susceptible to infection by Pratylenchus penetrans, the root lesion nematode. These migratory nematodes feed on roots and often physically disrupt the root tissues when many nematodes enter and move throughout the root to feed. The goal of this study was to find a root-specific or root-preferred promoter because of our interest in genetic engineering of lilies for nematode resistance. Several promoters, the Agrobacterium rolD and mas2, CaMV 35S, and rice RPC1 were examined in transgenic Lilium longiflorum, Easter lily, plants that contained the bar-uidA fusion gene under each promoter. Histochemical staining showed that the rolD, mas2, and CaMV 35S promoters directed gus gene expression in the cortex and stele of transformed roots. Gus expression was limited to the stele of roots with the rice RPC1 promoter making it unsuitable for engineering nematode resistance. The mas2 promoter was found to be expressed primarily in roots rather than shoots whereas the CaMV 35S promoter expressed well in both roots and shoots. Levels of gus specific activity were relatively high for both the mas2 and CaMV 35S promoter in roots but low with the rolD promoter. Because the mas2 promoter had relatively high levels of gus specific activity in roots and not shoots, and expression was throughout cortex and stele tissues of the roots, mas2 appears to be a promising promoter for engineering resistance to root lesion nematodes in Lilium longiflorum.