Imaging techniques for visualizing and analyzing precise morphology and gene expression patterns are essential for understanding biological processes during development in all organisms. With the aid of chemical screening, we developed a clearing method using chemical solutions, termed ClearSee, for deep imaging of morphology and gene expression in plant tissues. ClearSee rapidly diminishes chlorophyll autofluorescence while maintaining fluorescent protein stability. By adjusting the refractive index mismatch, whole-organ and whole-plant imaging can be performed by both confocal and two-photon excitation microscopy in ClearSee-treated samples. Moreover, ClearSee is applicable to multicolor imaging of fluorescent proteins to allow structural analysis of multiple gene expression. Given that ClearSee is compatible with staining by chemical dyes, the technique is useful for deep imaging in conjunction with genetic markers and for plant species not amenable to transgenic approaches. This method is useful for whole imaging for intact morphology and will help to accelerate the discovery of new phenomena in plant biological research.

Genetic incompatibility is a barrier contributing to species isolation and is caused by genetic interactions. We made a whole genome survey of two-way interacting loci acting within the gametophyte or zygote using independence tests of marker segregations in an F 2 population from an intersubspecific cross between O. sativa subspecies indica and japonica. We detected only one reproducible interaction, and identified paralogous hybrid incompatibility genes, DOPPELGANGER1 (DPL1) and DOPPELGANGER2 (DPL2), by positional cloning. Independent disruptions of DPL1 and DPL2 occurred in indica and japonica, respectively. DPLs encode highly conserved, plant-specific small proteins (∼10 kDa) and are highly expressed in mature anther. Pollen carrying two defective DPL alleles became nonfunctional and did not germinate, suggesting an essential role for DPLs in pollen germination. Although rice has many duplicated genes resulting from ancient whole genome duplication, the origin of this gene duplication was in recent small-scale gene duplication, occurring after Oryza-Brachypodium differentiation. Comparative analyses suggested the geographic and phylogenetic distribution of these two defective alleles, showing that loss-of-function mutations of DPL1 genes emerged multiple times in indica and its wild ancestor, O. rufipogon, and that the DPL2 gene defect is specific to japonica cultivars.Bateson-Dobzhansky-Muller incompatibility | reproductive isolation | speciation

Gene expression throughout the reproductive process in rice (Oryza sativa) beginning with primordia development through pollination/fertilization to zygote formation was analyzed. We analyzed 25 stages/organs of rice reproductive development including early microsporogenesis stages with 57,381 probe sets, and identified around 26,000 expressed probe sets in each stage. Fine dissection of 25 reproductive stages/organs combined with detailed microarray profiling revealed dramatic, coordinated and finely tuned changes in gene expression. A decrease in expressed genes in the pollen maturation process was observed in a similar way with Arabidopsis and maize. An almost equal number of ab initio predicted genes and cloned genes which appeared or disappeared coordinated with developmental stage progression. A large number of organ-/stage-specific genes were identified; notably 2,593 probe sets for developing anther, including 932 probe sets corresponding to ab initio predicted genes. Analysis of cell cycle-related genes revealed that several cyclin-dependent kinases (CDKs), cyclins and components of SCF E3 ubiquitin ligase complexes were expressed specifically in reproductive organs. Cell wall biosynthesis or degradation protein genes and transcription factor genes expressed specifically in reproductive stages were also newly identified. Rice genes homologous to reproduction-related genes in other plants showed expression profiles both consistent and inconsistent with their predicted functions. The rice reproductive expression atlas is likely to be the most extensive and most comprehensive data set available, indispensable for unraveling functions of many specific genes in plant reproductive processes that have not yet been thoroughly analyzed.