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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.

The blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.

Arabidopsis cryptochrome 2 (CRY2) is a blue light receptor that mediates light inhibition of hypocotyl elongation and long-day promotion of floral initiation. CRY2 is known to undergo blue light-dependent phosphorylation, which is believed to serve regulatory roles in the function of CRY2. We report here on a biochemical and genetics study of CRY2 phosphorylation. Using mass spectrometry analysis, we identified three serine residues in the CCE domain of CRY2 (S598, S599, and S605) that undergo blue light-dependent phosphorylation in Arabidopsis seedlings. A study of serine-substitution mutations in the CCE domain of CRY2 demonstrates that CRY2 contains two types of phosphorylation in the CCE domain, one in the serine cluster that causes electrophoretic mobility upshift and the other outside the serine cluster that does not seem to cause mobility upshift. We showed that mutations in the serine residues within and outside the serine cluster diminished blue light-dependent CRY2 phosphorylation, degradation, and physiological activities. These results support the hypothesis that blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.

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.

AtWNK9 is regulated by ABA and dehydration and is involved in drought tolerance in Arabidopsis.

WNK (with no lysine [K]) kinases play important regulatory roles in flowering, as well as salt and osmotic stress tolerance in plants. Here, we report that AtWNK9, a member of the Arabidopsis WNK gene family, was induced by exogenous abscisic acid (ABA) treatment and dehydration stress. Overexpression of AtWNK9 from the cauliflower mosaic virus 35S promoter in Arabidopsis resulted in increased sensitivity to ABA, strong inhibition of primary root elongation, increased proline accumulation, reduced stomatal aperture, and a reduced rate of water loss. In addition, plant survival under drought stress was improved compared to wild type. In contrast, a mutant with a T-DNA insertion in AtWNK9 showed reduced ABA sensitivity and an increased rate of water loss; further, it showed increased susceptibility to drought stress. The transcription of a number of ABA signaling components, including ABI1, ERA1, ABI3, and ABF3, was up-regulated in AtWNK9 transgenic plants and down-regulated in the wnk9 mutant in response to ABA. Some ABA-responsive and biosynthetic genes, as well as other drought-related genes, were altered at various levels in AtWNK9 transgenic plants and wnk9 mutants under dehydration stress. Overall, these findings suggest that AtWNK9 plays a positive role in ABA signaling and improves drought tolerance in transgenic Arabidopsis.

Heterologous Expression of a Gibberellin 2-Oxidase Gene from Arabidopsis thaliana Enhanced the Photosynthesis Capacity in Brassica napus L.

Gibberellins (GAs) are endogenous hormones that play an important role in regulating plant stature by increasing cell division and promoting seed germination. 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 reduced the plant height. As anticipated, the transgenic plant exhibited dwarf phenotype. Importantly, compared with the wild type, the transgenic plants had delayed the seed germination, increased the chlorophyll content (28.7–36.3%) and photosynthesis capacity (14.3–18.7%) in a single leaf. At the same time, the photosynthesis capacity of the whole plants was significantly enhanced (35.7–48.6%) due to the extra leaves and branches.

Construction and validation of a dual-transgene vector system for stable transformation in plants

In this study, we constructed dual-transgene vectors (pDT1, pDT7, and pDT7G) that simultaneously co-expressed two genes in plants. ACTIN2 and UBQ10 promoters were used to control the expression of these two genes. The 4×Myc, 3×HA, and 3×Flag reporter genes allowed for the convenient identification of a tunable co-expression system in plants, whereas the dexamethasone (Dex) inducible reporter gene C-terminus of the glucocorticoid receptor (cGR) provided Dex-dependent translocation of the fusion gene between the nucleus and cytoplasm. The function of pDT vectors was validated using four pairwise genes in Nicotiana benthamiana or Arabidopsis thaliana. The co-expression efficiency of two genes from the pDT1 and pDT7G vectors was 35% and 42%, respectively, which ensured the generation of sufficient transgenic materials. These pDT vectors are simple, reliable, efficient, and time-saving tools for the co-expression of two genes through a single transformation event and can be used in the study of protein-protein interactions or multi-component complexes.

Ectopic expression of GA 2-oxidase 6 from rapeseed (Brassica napus L.) causes dwarfism, late flowering and enhanced chlorophyll accumulation in Arabidopsis thaliana.

Gibberellins (GAs) are endogenous hormones that play an important role in higher plant growth and development. GA2-oxidase (GA2ox) promotes catabolism and inactivation of bioactive GAs or their precursors. In this study, we identified the GA2-oxidase gene, BnGA2ox6, and found it to be highly expressed in the silique and flower. Overexpression of BnGA2ox6 in Arabidopsis resulted in GA-deficiency symptoms, including inhibited elongation of the hypocotyl and stem, delayed seed germination, and late flowering. BnGA2ox6 overexpression reduced silique growth, but had no effect on seed development. Additionally, BnGA2ox6 overexpression enhanced chlorophyll b and total chlorophyll accumulation, and downregulated mRNA expression levels of the CHL1 and RCCR genes, which are involved in the chlorophyll degradation. These findings suggest that BnGA2ox6 regulates plant hight, silique development, flowering and chlorophyll accumulation in transgenic Arabidopsis.

F-box gene FOA2 regulates GA- and ABA- mediated seed germination in Arabidopsis

GA and ABA antagonize each other in controlling seed germination, but the molecular mechanism is not fully understood. The F-box proteins act as the most important SCF (SKP1, cullin/CDC53, F-box protein) complex subunit of the ubiquitin (Ub)-26S proteasome system, mediate diverse physiological processes ranging from hormonal signaling cascades to environmental stress responses (Santner and Estelle, 2010). We previously demonstrated that FOA1 (F-box overexpressed/oppressed ABA signaling) is an ABA signaling-related gene, and plays a negative role in ABA signaling (Peng et al., 2012). Our preliminary results showed that F-box protein (AT3G16740), homologous to FOA1, named as FOA2, is highly expressed in silique (Duan et al., 2013), suggesting it may be involved in seed development. In this study, we examined the expression patterns of FOA2 during seed maturation and imbibition. Interestingly, with the maturity of the seeds the transcript level of FOA2 increased, and reached plateau in the 27 DPA (days post-anthesis) silique (Figure 1A), whereas gradually decreased during seed imbibition (Figure 1B). Therefore, we postulated that FOA2 might function in regulating seed dormancy and germination. To address this hypothesis, we identified T-DNA insertion mutant (SALK_148443) foa2 and generated FOA2 transgenic lines. The RT-PCR and qPCR results confirmed that the foa2 is a null mutant, and the FOA2 was overexpressed in transgenic lines (Figure S1A–D, Tables S1 and S2 in Supporting Information). Overexpression of FOA2 delays seed germination (Figure S1F in Supporting Information), but the foa2 mutant showed no difference compared to the wild type (Figure S1E in Supporting Information), which may be a result of functional redundancy with other homologous proteins. Interestingly, in the presence of 10 4 mol L 1 GA 3 , the germination rate of FOA2 transgenic lines restored to the wild type (Figure S2 in Supporting Information). Moreover, in the presence of 10 2 μmol L 1 GA biosynthesis inhibitor paclobutrazol (PAC), the seed germination rate of transgen-ic lines was reduced by about 75%, 70%, and 62.5%, respectively , whereas only 25% in the wild-type Col-4 (Figure 1C and D). However, the seed germination of foa2 mutant reduced by 67%, while the wild-type Col-0 reduced by 80% (Figure 1C and E), indicating that overexpression of FOA2 results in increased PAC sensitive phenotype, and foa2 mutant is more resistant to PAC during seed germination. Moreover, the transcription of FOA2 was up-regulated by GA treatment during seed imbibition (Figure S3 in Supporting Information). These findings suggesting a negative role …

A photo-responsive F-box protein FOF2 regulates floral initiation by promoting FLC expression in Arabidopsis

Floral initiation is regulated by various genetic pathways in response to light, temperature, hormones and developmental status; however, the molecular mechanisms underlying the interactions between different genetic pathways are not fully understood. Here, we show that the photoresponsive gene FOF2 (F-box of flowering 2) negatively regulates flowering. FOF2 encodes a putative F-box protein that interacts specifically with ASK14, and its overexpression results in later flowering under both long-day and short-day photoperiods. Conversely, transgenic plants expressing the F-box domain deletion mutant of FOF2 (FOF2ΔF), or double loss of function mutant of FOF2 and FOL1 (FOF2-LIKE 1) present early flowering phenotypes. The late flowering phenotype of the FOF2 overexpression lines is suppressed by the flc-3 loss-of-function mutation. Furthermore, FOF2 mRNA expression is regulated by autonomous pathway gene FCA, and the repressive effect of FOF2 in flowering can be overcome by vernalization. Interestingly, FOF2 expression is regulated by light. The protein level of FOF2 accumulates in response to light, whereas it is degraded under dark conditions via the 26S proteasome pathway. Our findings suggest a possible mechanistic link between light conditions and the autonomous floral promotion pathway in Arabidopsis.