Reporters have been widely used to visualize gene expression, protein localization, and other cellular activities, but the commonly used reporters require special equipment, expensive chemicals, or invasive treatments. Here, we construct a new reporter RUBY that converts tyrosine to vividly red betalain, which is clearly visible to naked eyes without the need of using special equipment or chemical treatments. We show that RUBY can be used to noninvasively monitor gene expression in plants. Furthermore, we show that RUBY is an effective selection marker for transformation events in both rice and Arabidopsis. The new reporter will be especially useful for monitoring cellular activities in large crop plants such as a fruit tree under field conditions and for observing transformation and gene expression in tissue culture under sterile conditions.

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Pollen hydration is a prerequisite for pollen germination and subsequent pollen tube growth. As a result of two decades of biochemical studies and genetic identification of the eceriferum (cer) male-sterile mutants, the very-long-chain fatty acid (VLCFA) lipids in the pollen coat have been well known to play a critical role in pollen hydration (Preuss et al., 1993;Fiebig et al., 2000;Hiscock and Allen, 2008). VLCFAs are referred to as fatty acids with 20 or more carbons, which are synthesized through four sequential reactions catalyzed by 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase, and enoyl reductase (ECR). Among all the reactions, KCS-mediated condensation is the rate-limiting step and determines the length of the final acyl-CoA products (Haslam and Kunst, 2013). KCS6 (also known as CER6, POP1, and CUT1) catalyzes the production of C28 fatty acyl-CoAs and is essential for pollen hydration (Fiebig et al., 2000;Haslam and Kunst, 2013). In addition, BAHD acyltransferase CER2/CER2-like proteins are required for VLCFAs synthesis (Xia et al., 1997;Haslam et al., 2012Haslam et al., , 2015. It was proposed that CER2/CER2-like proteins function as novel components of the VLCFA elongation machinery through association with the specific KCS enzyme (Haslam and Kunst, 2013). CER2 acts redundantly with CER2-like2 (CER2L2) to affect male fertility under low-humidity environments (Haslam et al., 2015), indicating that CER2 and CER2L2 are likely involved in pollen hydration. However, the cytological and genetic evidence of CER2 and CER2L2 in male sterility remains elusive.

DNA methylation is an important epigenetic mark that regulates the expression of genes and transposons. RNA-directed DNA methylation (RdDM) is the main molecular pathway responsible for de novo DNA methylation in plants. Although the mechanism of RdDM has been well studied in Arabidopsis (Arabidopsis thaliana), most mutations in RdDM genes cause no remarkable developmental defects in Arabidopsis. Here, we isolated and cloned Five Elements Mountain 1 (FEM1), which encodes RNA-dependent RNA polymerase 2 (OsRDR2) in rice (Oryza sativa). Mutation in OsRDR2 abolished the accumulation of 24-nt small interfering RNAs, and consequently substantially decreased genome-wide CHH (H = A, C, or T) methylation. Moreover, male and female reproductive development was disturbed, which led to sterility in osrdr2 mutants. We discovered that OsRDR2-dependent DNA methylation may regulate the expression of multiple key genes involved in stamen development, meiosis, and pollen viability. In wild-type (WT) plants but not in osrdr2 mutants, genome-wide CHH methylation levels were greater in panicles, stamens, and pistils than in seedlings. The global increase of CHH methylation in reproductive organs of the WT was mainly explained by the enhancement of RdDM activity, which includes OsRDR2 activity. Our results, which revealed a global increase in CHH methylation through enhancement of RdDM activity in reproductive organs, suggest a crucial role for OsRDR2 in the sexual reproduction of rice.

SUMMARYInositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2a and AtIPK2b) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2a and AtIPK2b revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2a and AtIPK2b act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock-out mutants of atipk2a and atipk2b were indistinguishable from the wild type, whereas the atipk2a atipk2b double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2a and AtIPK2b resulted in severely reduced transmission of male gametophyte as a result of abnormal pollen development and defective pollen tube guidance. In addition, the early embryo development of the atipk2a atipk2b double mutant was also aborted. Expressing either catalytically inactive or substrate specificity-altered variants of AtIPK2b could not rescue the male gametophyte and embryogenesis defects of the atipk2a atipk2b double mutant, implying that the kinase activity of AtIPK2 is required for pollen development, pollen tube guidance and embryogenesis. Taken together, our results provide genetic evidence for the requirement of inositol polyphosphate signaling in plant sexual reproduction.