Rapid function analysis of OsiWAK1 using a Dual-Luciferase assay in rice.

In the past decade, the exploration of genetic resources in rice has significantly enhanced the efficacy of rice breeding. However, the exploration of genetic resources is hindered by the identification of candidate genes. To expedite the identification of candidate genes, this study examined tapetum programmed cell death-related genes OsiWAK1, OsPDT1, EAT1, TDR, and TIP2 to assess the efficacy of the Dual-Luciferase (Dual-LUC) assay in rapidly determining gene relationships. The study found that, in the Dual-LUC assay, OsiWAK1 and its various recombinant proteins exhibit comparable activation abilities on the EAT1 promoter, potentially indicating a false positive. However, the Dual-LUC assay can reveal that OsiWAK1 impacts both the function of its upstream regulatory factor OsPDT1 and the TDR/TIP2 transcription complex. By rapidly studying the relationship between diverse candidate genes and regulatory genes in a well-known trait via the Dual-LUC assay, this study provides a novel approach to expedite the determination of candidate genes such as genome-wide association study.

Full-length EFOP3 and EFOP4 proteins are essential for pollen intine development in Arabidopsis thaliana.

Pollen development is critical to plant reproduction, but the underlying regulatory molecular mechanisms have not been fully elucidated. The Arabidopsis (Arabidopsis thaliana) EFR3 OF PLANT 3 (EFOP3) and EFR3 OF PLANT 4 (EFOP4) genes encode members of the Armadillo (ARM) repeat superfamily that play key roles in pollen development. Herein, we demonstrate that EFOP3 and EFOP4 are co-expressed in pollen at anther stages 10-12, but loss-of-function of both EFOP3 and EFOP4 leads to male gametophyte sterility, irregular intine, and shriveled pollen grains at anther stage 12. We further established that full-length EFOP3 and EFOP4 specifically localize to the plasma membrane, and the integrity of these proteins is essential for pollen development. We observed uneven intine, less organized cellulose and reduced pectin content in mutant pollen compared with the wild-type. These, together with the misexpression of several genes related to cell wall metabolism in efop3-/- efop4+/- mutants, suggest that EFOP3 and EFOP4 may indirectly regulate the expression of these genes to affect intine formation, thus controlling Arabidopsis pollen fertility in a functionally redundant manner. Moreover, transcriptome analysis showed that the absence of EFOP3 and EFOP4 function affects multiple pollen development pathways. These results enhance our understanding of EFOPs proteins and their role in pollen development.

UDP-glucose epimerase 1, moonlighting as a transcriptional activator, is essential for tapetum degradation and male fertility in rice.

Multiple enzymes perform moonlighting functions distinct from their main roles. UDP-glucose epimerases (UGEs), a subclass of isomerases, catalyze the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). We identified a rice male-sterile mutant, osuge1, with delayed tapetum degradation and abortive pollen. The mutant osuge1 protein lacked UDP-glucose epimerase activity, resulting in higher UDP-Gal content and lower UDP-Glc levels in the osuge1 mutant compared with the wild type. Interestingly, we discovered that OsUGE1 participates in the TIP2/bHLH142-TDR-EAT1/DTD transcriptional regulatory cascade involved in tapetum degradation, in which TIP2 and TDR regulate the expression of OsUGE1 while OsUGE1 regulates the expression of EAT1. In addition, we found that OsUGE1 regulates the expression of its own gene by directly binding to an E-box element in the OsUGE1 promoter. Collectively, our results indicate that OsUGE1 not only functions as a UDP-glucose epimerase but also moonlights as a transcriptional activator to promote tapetum degradation, revealing a novel regulatory mechanism of rice reproductive development.

Oryza sativa PECTIN DEFECTIVE TAPETUM1 affects anther development through a pectin-mediated signaling pathway in rice.

Galacturonosyltransferase (GalAT) is required for the synthesis of pectin, an important component of plant cell walls that is also involved in signal transduction. Here, we describe the rice (Oryza sativa) male-sterile mutant O. sativa pectin-defective tapetum1 (ospdt1), in which GalAT is mutated. The ospdt1 mutant exhibited premature programmed cell death (PCD) of the tapetum and disordered pollen walls, resulting in aborted pollen grains. Pectin distribution in the anther sac was comparable between the mutant and the wild-type, suggesting that the structural pectin was not dramatically affected in ospdt1. Wall-associated kinases are necessary for the signal transduction of pectin, and the intracellular distribution of O. sativa indica WALL-ASSOCIATED KINASE1 (OsiWAK1), which binds pectic polysaccharides to its extracellular domain, was affected in ospdt1. OsiWAK1 RNA interference lines exhibited earlier tapetal PCD, similar to ospdt1. Furthermore, overexpression of OsiWAK1 in ospdt1 lines partially rescued the defects observed in ospdt1, suggesting that OsiWAK1 plays pivotal roles in the function of OsPDT1. These results suggest that the mutation of OsPDT1 does not dramatically affect structural pectin but affects components of the pectin-mediated signaling pathway, such as OsiWAK1, and causes male sterility.

OsMYB103 is essential for tapetum degradation in rice.

KEY MESSAGE: OsMYB103 positively regulates tapetum degradation, and functions downstream of TDR and upstream of EAT1 and PTC1. The precise regulation of programmed cell death (PCD) of the tapetum is crucial for the development of anthers and pollen in rice. In this study, we isolated and identified a male-sterile mutant of rice, osmyb103, which exhibited delayed tapetum degradation and defective mature pollen. Map-based cloning and genetic complementation revealed that OsMYB103 corresponded to the gene LOC_Os04g39470 and encoded a R2R3 MYB transcription factor. OsMYB103 was localized in the nucleus and was expressed preferentially in the tapetal cells and microspores of the anther. OsMYB103 regulated the expression of two transcription factors, ETERNAL TAPETUM 1 (EAT1) and PERSISTENT TAPETAL CELL 1 (PTC1), both of which regulated tapetum degradation positively. Moreover, the expression of OsMYB103 was directly regulated by the additional positive regulator of tapetum degradation TAPETUM DEGENERATION RETARDATION (TDR) and was able to interact with it. Genetic evidence confirmed that OsMYB103 acted upstream of EAT1. The results show that OsMYB103 is a positive regulator of tapetum degradation in rice. These findings provide a better understanding of the regulatory network that underlies degradation of the tapetum in rice.

Decreased Vascular Bundle 1 affects mitochondrial and plant development in rice.

BACKGROUND: Mitochondria are vital regulators of plant growth and development, constitute the predominant source of ATP, and participate in multiple anabolic and catabolic metabolic pathways. But the mechanism by which dysfunctional mitochondria affect plant growth remains unknown, and more mitochondria-defective mutants need to be identified. RESULTS: A mitochondria-defective mutant decreased vascular bundle 1 (dvb1) was isolated from rice mutant library mutagenized by EMS (ethylmethane sulfonate), which shows dwarfism, narrow leaves, short branches, few vascular bundles, and low fertility. Map-based cloning, genetic complementation, and phylogenetic analysis revealed that DVB1 encodes a structural protein classified in the Mic10 family and is required for the formation of cristae in mitochondria, and was primarily expressed in vascular bundles. The DVB1 protein is partially localized in the mitochondria and capable of forming dimers and polymers. Comparing with the wild type, disruption of amino acid metabolism and increased auxin synthesis were observed in dvb1 mutant which also showed increased sensitivity to the mitochondrial electron transport inhibitors. CONCLUSIONS: DVB1 belongs to Mic10 family and DVB1 is partially localized in the mitochondria. Further studies indicated that DVB1 is important for mitochondrial and plant development in rice.

Zebra leaf 15, a receptor-like protein kinase involved in moderate low temperature signaling pathway in rice.

BACKGROUND: Zebra leaf mutants are an important resource for studying leaf colour in rice. In most such mutants, the zebra leaf phenotype results from defective chloroplast biogenesis. The molecular mechanism by which zebra leaves develop remains unclear, so additional zebra-leaf mutants need to be identified. RESULTS: We isolated a novel rice zebra-leaf mutant, named zebra leaf 15 (z15), which showed transversely striped leaves with yellow-green or white-green sectors, in which chloroplast structure was disturbed. Transmission electron microscopy revealed that the structure of various organelles was impaired in yellow/white sectors. Z15, a single-copy gene in the rice genome, encodes a receptor-like protein kinase. Subcellular localization analysis indicates that Z15 and z15 are localized on the plasma membrane. The expression of Z15 is induced by moderate low temperature (18 °C). The mutation of Z15 influenced the expression of two downstream genes, OsWRKY71 and OsMYB4, that were responsive to moderate low temperature. The results show that Z15 plays a crucial role in the early stages of the response to moderate low temperature in rice. CONCLUSIONS: We identified a novel zebra-leaf mutant (z15) that impaired chloroplast structure in rice, LOC_Os05g12680, encoding a receptor-like protein kinase. Further study indiceted that Z15 plays a crucial role in the early stages of the response to moderate low temperature in rice.

Dwarf and short grain 1, encoding a putative U-box protein regulates cell division and elongation in rice.

Plant hormones coordinate a plant's responses to environmental stimuli and the endogenous developmental programs for cell division and elongation. Brassinosteroids are among the most important of these hormones in plant development. Recently, the ubiquitin-26S-proteasome system was identified to play a key role in hormone biology. In this study, we analyzed the function of a rice (Oryza sativa) gene, DSG1, which encodes a U-box E3 ubiquitin ligase. In the dsg1 mutant (an allelic mutant of tud1), the lengths of the roots, internodes, panicles, and seeds were shorter than that in the wild-type, which was due to defects in cell division and elongation. In addition, the leaves of the dsg1 mutant were wider and curled. The DSG1 protein is nuclear- and cytoplasm-localized and does not show tissue specificity in terms of its expression, which occurs in roots, culms, leaves, sheaths, and spikelets. The dsg1 mutant is less sensitive to brassinosteroid treatment than the wild-type, and DSG1 expression is negatively regulated by brassinosteroids, ethylene, auxin, and salicylic acid. These results demonstrate that DSG1 positively regulates cell division and elongation and may be involved in multiple hormone pathways.