Rice transcription factor GAMYB modulates bHLH142 and is homeostatically regulated by TDR during anther tapetal and pollen development.

GIBBERELLIN MYB GENE (GAMYB), UNDEVELOPED TAPETUM1 (UDT1), TDR INTERACTING PROTEIN2 (TIP2/bHLH142), TAPETUM DEGENERATION RETARDATION (TDR), and ETERNAL TAPETUM 1/DELAYED TAPETUM DEGENERATION (EAT1/DTD) are important transcription factors that play a crucial role during pollen development in rice. This study demonstrates that bHLH142 acts downstream of UDT1 and GAMYB and works as a 'hub' in these two pollen pathways. We show that GAMYB modulates bHLH142 expression through specific binding to the MYB motif of the bHLH142 promoter during the early stage of pollen development, while TDR acts as a transcriptional repressor of the GAMYB modulation of bHLH142 by binding to the E-box close to the MYB motif on the promoter. Altered expression of these transcription factors highlights that a tight, precise, and coordinated regulation among them is essential for normal pollen development. Most notably, we show that the regulatory pathways of GAMYB and UDT1 rely on bHLH142 in a direct and indirect manner, respectively, and function in different tissues with distinct biological roles during pollen development. This study advances our understanding of the molecular mechanisms of rice pollen development.

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice.

KEY MESSAGE: A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice. A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides - 351 and - 121, which contains the sugar response complex (- 266 to - 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.

Spike Activator 1, Encoding a bHLH, Mediates Axillary Bud Development and Spike Initiation in Phalaenopsis aphrodite.

Double-spikes Phalaenopsis orchids have greater market value than those with single-spike. In this study, a gene designated as Spike Activator 1 (SPK1), which encodes a basic helix-loop-helix (bHLH) transcription factor, was isolated and characterized from Phalaenopsis aphrodite (moth orchid). SPK1 was highly expressed in the meristematic tissues. In the axillary bud, SPK1 was highly upregulated by a moderately low temperature of 20 °C but downregulated by a spike inhibition temperature of 30 °C. SPK1 protein is localized in the nucleus. Another bHLH, bHLH35, which is also highly expressed in young tissues in the same way as SPK1 was also identified. In contrast to SPK1, bHLH35 transcripts are downregulated at 20 °C but upregulated at 30 °C. Bimolecular florescence complementation assay and yeast two-hybrid assays indicated that SPK1 interacts with bHLH35 and forms a heterodimer. Virus-induced gene silencing (VIGS) showed that 7 out of 15 vector control plants produced double spikes but that only 1 out of 15 VIGS-spk1 plants produced double spikes. RT-qPCR results indicated that VIGS-spk1 downregulated gene expression levels of SPK1, FT, CYCB, and EXPA8. Overall, we propose that SPK1 plays an essential role in early axillary bud development and spike initiation of P. aphrodite.

The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice.

Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

The promoter and the 5'-untranslated region of rice metallothionein OsMT2b gene are capable of directing high-level gene expression in germinated rice embryos.

KEY MESSAGE: Critical regions within the rice metallothionein OsMT2b gene promoter are identified and the 5'-untranslated region (5'-UTR) is found essential for the high-level promoter activity in germinated transgenic rice embryos. Many metallothionein (MT) genes are highly expressed in plant tissues. A rice subfamily p2 (type 2) MT gene, OsMT2b, has been shown previously to exhibit the most abundant gene expression in young rice seedling. In the present study, transient expression assays and a transgenic approach were employed to characterize the expression of the OsMT2b gene in rice. We found that the OsMT2b gene is strongly and differentially expressed in germinated rice embryos during seed germination and seedling development. Histochemical staining analysis of transgenic rice carrying OsMT2b::GUS chimeric gene showed that high-level GUS activity was detected in germinated embryos and at the meristematic part of other tissues during germination. Deletion analysis of the OsMT2b promoter revealed that the 5'-flanking region of the OsMT2b between nucleotides -351 and -121 relative to the transcriptional initiation site is important for promoter activity in rice embryos, and this region contains the consensus sequences of G box and TA box. Our study demonstrates that the 5'-untranslated region (5'-UTR) of OsMT2b gene is not only necessary for the OsMT2b promoter activity, but also sufficient to augment the activity of a minimal promoter in both transformed cell cultures and germinated transgenic embryos in rice. We also found that addition of the maize Ubi intron 1 significantly enhanced the OsMT2b promoter activity in rice embryos. Our studies reveal that OsMT2b351-ubi(In) promoter can be applied in plant transformation and represents potential for driving high-level production of foreign proteins in transgenic rice.

Tightly Controlled Expression of bHLH142 Is Essential for Timely Tapetal Programmed Cell Death and Pollen Development in Rice.

Male sterility is important for hybrid seed production. Pollen development is regulated by a complex network. We previously showed that knockout of bHLH142 in rice (Oryza sativa) causes pollen sterility by interrupting tapetal programmed cell death (PCD) and bHLH142 coordinates with TDR to modulate the expression of EAT1. In this study, we demonstrated that overexpression of bHLH142 (OE142) under the control of the ubiquitin promoter also leads to male sterility in rice by triggering the premature onset of PCD. Protein of bHLH142 was found to accumulate specifically in the OE142 anthers. Overexpression of bHLH142 induced early expression of several key regulatory transcription factors in pollen development. In particular, the upregulation of EAT1 at the early stage of pollen development promoted premature PCD in the OE142 anthers, while its downregulation at the late stage impaired pollen development by suppressing genes involved in pollen wall biosynthesis, ROS scavenging and PCD. Collectively, these events led to male sterility in OE142. Analyses of related mutants further revealed the hierarchy of the pollen development regulatory gene network. Thus, the findings of this study advance our understanding of the central role played by bHLH142 in the regulatory network leading to pollen development in rice and how overexpression of its expression affects pollen development. Exploitation of this novel functionality of bHLH142 may confer a big advantage to hybrid seed production.

Efficient and heritable transformation of Phalaenopsis orchids.

BACKGROUND: Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species. Phal. orchids have many unique biological features. However, investigation of these features and validation on their biological functions are limited due to the lack of an efficient transformation method. RESULTS: We developed a heritable and efficient Agrobacterium- mediated transformation using protocorms derived from tetraploid or diploid Phal. orchids. A T-DNA vector construct containing eGFP driven by ubiquitin promoter was subjected to transformation. An approximate 1.2-5.2 % transformation rate was achieved. Genomic PCR confirmed that hygromycin selection marker, HptII gene and target gene eGFP were integrated into the orchid genome. Southern blotting indicated a low T-DNA insertion number in the orchid genome of the transformants. Western blot confirmed the expression of eGFP protein in the transgenic orchids. Furthermore, the GFP signal was detected in the transgenic orchids under microscopy. After backcrossing the pollinia of the transgenic plants to four different Phal. orchid varieties, the BC1 progenies showed hygromycin resistance and all surviving BC1 seedlings were HptII positive in PCR and expressed GFP protein as shown by western blot. CONCLUSIONS: This study demonstrated a stable transformation system was generated for Phal. orchids. This useful transformation protocol enables functional genomics studies and molecular breeding.