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.

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.

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.

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples.

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning. This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 °C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.