Dongying Tang

Piercing and vacuum infiltration of the mature embryo: a simplified method for Agrobacterium-mediated transformation of indica rice

Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T0) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T0 plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation was further confirmed by the genetic and molecular analyses of T1 transformants.

A Lectin Receptor Kinase Positively Regulates ABA Response During Seed Germination and Is Involved in Salt and Osmotic Stress Response

Lectin receptor-like kinases (LecRK) are widespread in higher plants; however, little is known about their physiological roles. In this study, At1g70130 (designated LecRK-b2), an Arabidopsis LecRK gene, has been investigated. LecRK-b2 was predominantly expressed during seed germination, and its expression was ceased following germination. The expression of LecRK-b2 was induced by abscisic acid (ABA), salt, and osmotic stress. LecRK-b2 loss-of-function mutation slightly reduced the ABA sensitivity during seed germination, and this reduced sensitivity was demonstrated not due to lower ABA accumulation level in the seeds. Dual-luciferase transient expression assay confirmed that the transcription factor ABSCISIC ACID INSENSITIVE3 (ABI3) could activate the luciferase under driving of LecRK-b2 promoter. LecRK-b2 transcription level was found to be down-regulated in abi3 during seed germination. Furthermore, LecRK-b2 loss-of-function mutation reduced the salt and osmotic sensitivity during early development stage of Arabidopsis. Taken together, these results suggest that LecRK-b2 functions as a positive regulator of the ABA response during the seed germination and is involved in salt and osmotic stress response in the early development stage.

An Annexin V-based biosensor for quantitatively detecting early apoptotic cells

In this paper, we reported that a novel biosensor was developed to detect early apoptotic cells by the specific interaction between Annexin V and phosphatidylserine based on electrochemical impedance. Annexin V was immobilized on a self-assembled layer of gold nanoparticles, which allowed stable and high loading of Annexin V on the electrode surface, offering the possibility of sensitivity enhancement. Early apoptotic cells showed an increased exposition of phosphatidylserine on the cell membrane caused by physiological and pathological response reaction, leading to a strong interaction between the apoptotic cells and the electrode surface, which could be probed by electrochemical impedance spectroscopy. As examined using a model system of cells integrated by phosphatidylserine-modified liposome and a real one of early apoptotic cell induced by 5-fluorouracil, this biosensor demonstrated the great potential for rapid detection of cell apoptosis and drug screening. The results agreed well with those obtained using fluorescence microscopy and flow cytometry.

Molecular character of a phosphatase 2C (PP2C) gene relation to stress tolerance in Arabidopsis thaliana

Protein phosphatases type 2C (PP2Cs) from group A, which includes the ABI1/HAB1 and PP2CA branches, are key negative regulators of ABA signaling. HAI-1 gene had been shown to affect both seed and vegetative responses to ABA, which is one of PP2Cs clade A in Arabidopsis thaliana. Transgenic plants containing pHAI-1::GUS (β-glucuronidase) displayed GUS activity existing in the vascular system of leave veins, stems and petioles. Green fluorescent protein fused HAI-1 (HAI-1-GFP) was found in the nucleus through transient transformation assays with onion epidermal cells. The water-loss assays indicated the loss-of-function mutants did not show symptoms of wilting and they had still turgid green rosette leaves. The assays of seed germination by exogenous ABA and NaCl manifested that the loss-of-function mutants displayed higher insensitivity than wild-type plants. Taken together, the final results suggest that the HAI-1 (AT5G59220) encoded a nuclear protein and it can be highly induced by ABA and wound in Arabidposis, the stress-tolerance phenotype showed a slightly improvement when HAI-1 gene was disrupted.

Dwarfism in Brassica napus L. induced by the over-expression of a gibberellin 2-oxidase gene from Arabidopsis thaliana

Gibberellins (GAs) are endogenous hormones that play an important role in regulating plant stature by increasing cell division and elongation in stem internodes. The GA2-oxidase gene from Arabidopsis thaliana (AtGA2ox8) was introduced into Brassica napus L. by Agrobacterium-mediated floral-dip transformation with the aim of decreasing the amount of bioactive GA and hence reducing plant stature. As anticipated, the transgenic plants exhibited dwarf phenotype. Compared with the wild type, the transgenic plants had increased primary branches (by 14.1–15.3%) and siliques (by 10.8–15.2%), which resulted in a significant increase in the seed yield (by 9.6–12.4%). Moreover, the contents of anthocyanin in leaves of 60-day-old transgenic plants was about 9.4-fold higher in winter and about 6.8-fold higher in summer than the wild type. These excellent agronomic traits of the transgenic plants could not only improve the lodging resistance and seed yields, but also protect them against stress. Therefore, the over-expression of AtGA2ox8 might be used to produce dwarf varieties and increase seed yield in Brassica napus L.

DBB1a, involved in gibberellin homeostasis, functions as a negative regulator of blue light-mediated hypocotyl elongation in Arabidopsis

Double B-box 1a (DBB1a) belongs to the zinc-finger family proteins in Arabidopsis thaliana. Transcriptional analysis uncovered that the DBB1a gene expression was blue light-dependently regulated, and the transcript level of DBB1a in cry1cry2 was decreased but not in phyAphyB compared to wild type under blue light conditions. Transgenic plants containing pDBB1a:GUS (β-glucuronidase) displayed GUS activity in the vascular system of leaves and petioles. Green fluorescent protein (GFP)-fused DDB1a (DBB1a-GFP) protein was found in the nucleus in transient transformation assays with onion epidermal cells as well as in stable transgenic Arabidopsis plants. To investigate the function of DBB1a, we generated DBB1a over-expressing and under-expressing transgenic Arabidopsis plants. Analysis of hypocotyl growth of these lines indicated that DBB1a promoted hypocotyl elongation under blue light condition. The phenotype of transgenic plants with DBB1a over-expression could be impaired by a gibberellin (GA)-biosynthesis inhibitor. Moreover, the expression analysis of GA metabolic and catabolic genes in DBB1a transgenic lines indicated that the DBB1a suppressed GA2-oxidase1 (GA2ox1) and GA2-oxidase8 (GA2ox8) expression, but induced GA3β-hydroxygenase1 (GA3ox1) and GA20-oxidase1 (GA20ox1) expression under blue light. Taken together, we concluded that DBB1a promotes hypocotyl elongation under blue light condition through an increase in bioactive GA levels in Arabidopsis.

Arabidopsis F-box gene FOA1 involved in ABA signaling

The expression of FOA1 (F-box overexpressed/oppressed ABA signaling) in different organs of Arabidopsis, and in response to ABA and NaCl, was analyzed. The expression level of FOA1 is higher in the root and is lower in the stem, and is induced rapidly by ABA and NaCl. The phenotypes of T-DNA insertion mutant foa1 and FOA1 overexpression lines FOA1ox1 and FOA1ox2 were analyzed. The foa1 mutant exhibited a lower germination rate, shorter root length, more stomatal opening, increased proline accumulation and hypersensitivity to ABA compared with the wild type. In contrast, the overexpression lines showed lower sensitivity to ABA than the wild type. The expression levels of several ABA and stress-responsive transcription factors and genes were altered in the foa1 mutant in response to ABA. Compared with the wild type, the expression levels of ABA-responsive transcription factors were higher, but ABA and stress-responsive genes were lower in foa1 mutant. This study demonstrates that FOA1 is an ABA signaling-related gene, and may play a negative role in ABA signaling.

Regulation of salt and ABA responses by CIPK14, a calcium sensor interacting protein kinase in Arabidopsis

Calcium and protein kinase serve as the common mediators to regulate plant responses to multiple stresses including salt and ABA stimulus. Here we reported a novel protein kinase (CIPK14) that regulated the responses to ABA treatment and salt stress in Arabidopsis. CIPK14 transcripts, capable been checked in roots, stems, leaves and flowers, were highly expressed in flowers and roots. CIPK14 was induced by ABA and salt treatments. The disruption of CIPK14 altered the transcriptional pattern of a gene marker line related to ABA and salt responses, and the results suggested that CIPK14 probably was responsible to the control of the salt and ABA responses. Comparing with wild types, the lines inserted with the T-DNA in which CIPK14 gene expression was knocked out were also more sensitive to ABA and salt stimulus, showing low germination rate and the less root elongation. While, when these conditioned seeds were treated with norflurazon, their germination percentages could recover to a certain extent. We also found that exogenous calcium could have an effect on the transcription of CIPK14 under ABA and salt treatments, and it seemed that calcium ion might work upstream CIPK14 to regulate the plant response to ABA and salt response.

A Defect in Zinc Finger Protein Double B-box 1a (DBB1a) Causes Abnormal Floral Development in Arabidopsis

The double B-box (DBB) type zinc finger protein has thus far been shown to be involved in photomorphegenesis in Arabidopsis thaliana. Here, we show that DBB1a is expressed in the embryo, cytolden, and flower. Misexpression of DBB1a in mutant plants resulted in abnormal numbers and patterns of floral organs. We further show that DBB1a could regulate expression of several floral homeotic genes, including APETALA 2, APETALA 3, PISTILLATA, and AGAMOUS. Interestingly, expression of the microRNA gene MiR172, which is involved in organ boundary establishment, was also misregulated in the dbb1a mutant plants. Our study identified a previously uncharacterized role of DDB1a in regulation of expression of floral homeotic genes and miR172, which is important for understanding of floral pattern formation.

Over-expression of the AtGA2ox8 gene decreases the biomass accumulation and lignification in rapeseed (Brassica napus L.).

Gibberellin 2-oxidase (GA 2-oxidase) plays very important roles in plant growth and development. In this study, the AtGA2ox8 gene, derived from Arabidopsis (Arabidopsis thaliana), was transformed and over-expressed in rapeseed (Brassica napus L.) to assess the role of AtGA2ox8 in biomass accumulation and lignification in plants. The transgenic plants, identified by resistant selection, polymerase chain reaction (PCR) and reverse-transcription PCR (RT-PCR) analyses, and green fluorescence examination, showed growth retardation, flowering delay, and dwarf stature. The fresh weight and dry weight in transgenic lines were about 21% and 29% lower than those in wild type (WT), respectively, and the fresh to dry weight ratios were higher than that of WT. Quantitative measurements demonstrated that the lignin content in transgenic lines decreased by 10%–20%, and histochemical staining results also showed reduced lignification in transgenic lines. Quantitative real-time PCR analysis indicated that the transcript levels of lignin biosynthetic genes in transgenic lines were markedly decreased and were consistent with the reduced lignification. These results suggest that the reduced biomass accumulation and lignification in the AtGA2ox8 overexpression rapeseed might be due to altered lignin biosynthetic gene expression.