Rice ILI atypical bHLH transcription factors antagonize OsbHLH157/OsbHLH158 during brassinosteroid signaling.

Brassinosteroids (BRs) are a group of steroid hormones that play crucial roles in plant growth and development. Atypical bHLH transcription factors that lack the basic region for DNA binding have been implicated in BR signaling. However, the underlying mechanisms of atypical bHLHs in regulation of rice (Oryza sativa) BR signaling are still largely unknown. Here, we describe a systematic characterization of INCREASED LEAF INCLINATION (ILI) subfamily atypical bHLH transcription factors in rice. A total of 8 members, ILI1 to ILI8, with substantial sequence similarity were retrieved. Knockout and overexpression analyses demonstrated that these ILIs play unequally redundant and indispensable roles in BR-mediated growth and development in rice, with a more prominent role for ILI4 and ILI5. The ili3/4/5/8 quadruple and ili1/3/4/7/8 quintuple mutants displayed tremendous BR-related defects with severe dwarfism, erect leaves, and sterility. Biochemical analysis showed that ILIs interact with OsbHLH157 and OsbHLH158, which are also atypical bHLHs and have no obvious transcriptional activity. Overexpression of OsbHLH157 and OsbHLH158 led to drastic BR-defective growth, whereas the osbhlh157 osbhlh158 double mutant developed a typical BR-enhanced phenotype, indicating that OsbHLH157 and OsbHLH158 play a major negative role in rice BR signaling. Further transcriptome analyses revealed opposite effects of ILIs and OsbHLH157/OsbHLH158 in regulation of downstream gene expression, supporting the antagonism of ILIs and OsbHLH157/OsbHLH158 in maintaining the balance of BR signaling. Our results provide insights into the mechanism of BR signaling and plant architecture formation in rice.

OsTKPR2 is part of a sporopollenin-producing metabolon required for exine formation in rice.

The sporopollenin polymer is a major component of the pollen exine. Fatty acid derivatives synthesized in the tapetum are among the precursors of sporopollenin. Progress has been made to understand sporopollenin metabolism in rice; however, the underlying molecular mechanisms remain elusive. We found that OsTKPR2 and OsTKPR1 share a similar expression pattern, and their coding proteins have a similar subcellular localization and enzyme activities towards reduced tetraketide α-pyrone and hydroxylated tetraketide α-pyrone. Unexpectedly, OsTKPR1pro:OsTKPR2-eGFP could not rescue the phenotype of ostkpr1-4. Three independent ostkpr2 mutant lines generated by CRISPR/Cas9 displayed reduced male fertility to various extents which were correlated with the severity of gene disruptions. Notably, the anther cuticle, Ubisch bodies, and pollen development were affected in the ostkpr2-1 mutant, where a thinner pollen exine was noticed. OsTKPR1 and OsTKPR2 were integrated into a metabolon including OsACOS12 and OsPKS2, which resulted in a significant increased enzymatic efficiency when both OsTKPR1 and OsTKPR2 were present, indicating the mutual dependence of OsTKPR2 and OsTKPR1 for their full biochemical activities. Thus, our results demonstrated that OsTKPR2 is required for anther and pollen development where an OsTKPR2-containing metabolon is functional during rice sporopollenin synthesis. Furthermore, the cooperation and possible functional divergence between OsTKPR2 and OsTKPR1 is also discussed.

OsCPD1 and OsCPD2 are functional brassinosteroid biosynthesis genes in rice.

CONSTITUTIVE PHOTOMORPHOGENIC DWARF (CPD), member of the CYP90A family of cytochrome P450 (CYP450) monooxygenase, is an essential component of brassinosteroids (BRs) biosynthesis pathway. Compared with a single CPD/CYP90A1 in Arabidopsis thaliana, two highly homologous CPD genes, OsCPD1/CYP90A3 and OsCPD2/CYP90A4, are present in rice genome. There is still no genetic evidence so far about the requirement of OsCPD1 and OsCPD2 in rice BR biosynthesis. In this study, we reported the functional characterization of OsCPD genes using CRISPR/Cas9 gene editing technology. The overall growth and development of oscpd1 and oscpd2 single knock-out mutants was indistinguishable from the wild-type, whereas, the oscpd1 oscpd2 double mutant displayed multiple and obvious BR-related defects. Cytological analyses further indicated the defective cell elongation in oscpd1 oscpd2 double mutant. The oscpd double mutants had a lower endogenous BR level and could be restored by the application of the brassinolide (BL). Moreover, overexpression of OsCPD1 and OsCPD2 led to a typical BR enhanced phenotype, with enlarged leaf angle and increased grain size. Taken together, our results provide direct genetic evidence that OsCPD1 and OsCPD2 play essential and redundant roles in maintenance of plant architecture by modulating BR biosynthesis in rice.

Reinforcement of CHH methylation through RNA-directed DNA methylation ensures sexual reproduction in rice.

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.

Tapetal 3-Ketoacyl-Coenzyme A Synthases Are Involved in Pollen Coat Lipid Accumulation for Pollen-Stigma Interaction in Arabidopsis.

Pollen coat lipids form an outer barrier to protect pollen itself and play essential roles in pollen-stigma interaction. However, the precise molecular mechanisms underlying the production, deposition, regulation, and function of pollen coat lipids during anther development remain largely elusive. In lipid metabolism, 3-ketoacyl-coenzyme A synthases (KCS) are involved in fatty acid elongation or very-long-chain fatty acid (VLCFA) synthesis. In this study, we identified six members of the Arabidopsis KCS family expressed in anther. Among them, KCS7, KCS15, and KCS21 were expressed in tapetal cells at anther stages 8-10. Further analysis demonstrated that they act downstream of male sterility 1 (MS1), a regulator of late tapetum development. The kcs7/15/21 triple mutant is fertile. Both cellular observation and lipid staining showed pollen coat lipid was decreased in kcs7/15/21 triple mutant. After landing on stigma, the wild-type pollen grains were hydrated for about 5 min while the kcs7/15/21 triple mutant pollen took about 10 min to hydrate. Pollen tube growth of the triple mutant was also delayed. These results demonstrate that the tapetum-localized KCS proteins are involved in the accumulation of pollen coat lipid and reveal the roles of tapetal-derived pollen coat lipid for pollen-stigma interaction.

A reporter for noninvasively monitoring gene expression and plant transformation.

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.

Phenylpropanoid Derivatives Are Essential Components of Sporopollenin in Vascular Plants.

The outer wall of pollen and spores, namely the exine, is composed of sporopollenin, which is highly resistant to chemical reagents and enzymes. In this study, we demonstrated that phenylpropanoid pathway derivatives are essential components of sporopollenin in seed plants. Spectral analyses showed that the autofluorescence of Lilium and Arabidopsis sporopollenin is similar to that of lignin. Thioacidolysis and NMR analyses of pollen from Lilium and Cryptomeria further revealed that the sporopollenin of seed plants contains phenylpropanoid derivatives, including p-hydroxybenzoate (p-BA), p-coumarate (p-CA), ferulate (FA), and lignin guaiacyl (G) units. The phenylpropanoid pathway is expressed in the tapetum in Arabidopsis, consistent with the fact that the sporopollenin precursor originates from the tapetum. Further germination and comet assays showed that this pathway plays an important role in protection of pollen against UV radiation. In the pteridophyte plant species Ophioglossum vulgatum and Lycopodium clavata, phenylpropanoid derivatives including p-BA and p-CA were also detected, but G units were not. Taken together, our results indicate that phenylpropanoid derivatives are essential for sporopollenin synthesis in vascular plants. In addition, sporopollenin autofluorescence spectra of bryophytes, such as Physcomitrella and Haplocladium, exhibit distinct characteristics compared with those of vascular plants, indicating the diversity of sporopollenin among land plants.

Cytoplasmic ribosomal protein L14B is essential for fertilization in Arabidopsis.

Plant cytoplasmic ribosomal proteins not only participate in protein synthesis, but also have specific roles in developmental regulation. However, the high heterogeneity of plant ribosome makes our understanding of these proteins very limited. Here we reported that RPL14B, a component of the ribosome large subunit, is critical for fertilization in Arabidopsis. RPL14B is existed in a majority of organs and tissues. No homozygous rpl14b mutant is available, indicating that RPL14B is irreplaceable for sexual reproduction. Smaller-sized rpl14b pollens could germinate normally, but pollen tube competitiveness is grievously weakened. Beside, cell fate specification is impaired in female gametophytes from heterozygous rpl14b/RPL14B ovules, resulting in defect of micropylar pollen tube attraction. However, this defect could be restored by restricted expression of RPL14B in synergid cells. Successful fertilization requires normal pollen tube growth and precise pollen tube guidance. Thus our results show a novel role of RPL14B in fertilization and shed new light on regulatory mechanism of pollen tube growth and precise pollen tube guidance.

Repurposing of Anthocyanin Biosynthesis for Plant Transformation and Genome Editing.

CRISPR/Cas9 gene editing technology has been very effective in editing genes in many plant species including rice. Here we further improve the current CRISPR/Cas9 gene editing technology in both efficiency and time needed for isolation of transgene-free and target gene-edited plants. We coupled the CRISPR/Cas9 cassette with a unit that activates anthocyanin biosynthesis, providing a visible marker for detecting the presence of transgenes. The anthocyanin-marker assisted CRISPR (AAC) technology enables us to identify transgenic events even at calli stage, to select transformants with elevated Cas9 expression, and to identify transgene-free plants in the field. We used the AAC technology to edit LAZY1 and G1 and successfully generated many transgene-free and target gene-edited plants at T1 generation. The AAC technology greatly reduced the labor, time, and costs needed for editing target genes in rice.

Identification of AtHsp90.6 involved in early embryogenesis and its structure prediction by molecular dynamics simulations.

Heat-shock protein of 90 kDa (Hsp90) is a key molecular chaperone involved in folding the synthesized protein and controlling protein quality. Conformational dynamics coupled to ATPase activity in N-terminal domain is essential for Hsp90's function. However, the relevant process is still largely unknown in plant Hsp90s, especially those required for plant embryogenesis which is inextricably tied up with human survival. Here, AtHsp90.6, a member of Hsp90 family in Arabidopsis, was firstly identified as a protein essential for embryogenesis. Thus we modelled AtHsp90.6 in its functionally closed 'lid-down' and open 'lid-up' states, exploring the nucleotide binding mechanism in these two states. Free energy landscape and electrostatic potential analysis revealed the switching mechanism between these two states. Collectively, this study quantitatively analysed the conformational changes of AtHsp90.6 bound to ATP or ADP. This result may help us understand the mechanism of action of AtHsp90.6 in future.

Enzyme activities of Arabidopsis inositol polyphosphate kinases AtIPK2α and AtIPK2ß are involved in pollen development, pollen tube guidance and embryogenesis.

Inositol polyphosphate kinase (IPK2) is a key component of inositol polyphosphate signaling. There are two highly homologous inositol polyphosphate kinases (AtIPK2α and AtIPK2ß) in Arabidopsis. Previous studies that overexpressed or reduced the expression of AtIPK2α and AtIPK2ß revealed their roles in auxiliary shoot branching, abiotic stress responses and root growth. Here, we report that AtIPK2α and AtIPK2ß act redundantly during pollen development, pollen tube guidance and embryogenesis. Single knock-out mutants of atipk2α and atipk2ß were indistinguishable from the wild type, whereas the atipk2α atipk2ß double mutant could not be obtained. Detailed genetic and cytological investigations showed that the mutation of AtIPK2α and AtIPK2ß 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 atipk2α atipk2ß double mutant was also aborted. Expressing either catalytically inactive or substrate specificity-altered variants of AtIPK2ß could not rescue the male gametophyte and embryogenesis defects of the atipk2α atipk2ß 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.