Arabidopsis HOT3/eIF5B1 constrains rRNA RNAi by facilitating 18S rRNA maturation.

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3' end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3' end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3' end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3' end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3' ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3' portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.

HEI10 is subject to phase separation and mediates RPA1a degradation during meiotic interference-sensitive crossover formation.

Reciprocal exchanges of DNA between homologous chromosomes during meiosis, or crossovers (COs), shuffle genetic information in gametes and progeny. In many eukaryotes, the majority of COs (class I COs) are sensitive to a phenomenon called interference, which influences the occurrence of closely spaced double COs. Class I COs depend on a group of factors called ZMM (Zip, Msh, Mer) proteins including HEI10 (Human Enhancer of Invasion-10). However, how these proteins are recruited to class I CO sites is unclear. Here, we show that HEI10 forms foci on chromatin via a liquid-liquid phase separation (LLPS) mechanism that relies on residue Ser70. A HEI10S70F allele results in LLPS failure and a defect in class I CO formation. We further used immunoprecipitation-mass spectrometry to identify RPA1a (Replication Protein A 1) as a HEI10 interacting protein. Surprisingly, we find that RPA1a also undergoes phase separation and its ubiquitination and degradation are directly regulated by HEI10. We also show that HEI10 is required for the condensation of other class I CO factors. Thus, our results provide mechanistic insight into how meiotic class I CO formation is controlled by HEI10 coupling LLPS and ubiquitination.

DNA polymerase epsilon interacts with SUVH2/9 to repress the expression of genes associated with meiotic DSB hotspot in Arabidopsis.

Meiotic recombination is initiated by the SPORULATION 11 (SPO11)-triggered formation of double-strand breaks (DSBs) that usually occur in open chromatin with active transcriptional features in many eukaryotes. However, gene transcription at DSB sites appears to be detrimental for repair, but the regulatory mechanisms governing transcription at meiotic DSB sites are largely undefined in plants. Here, we demonstrate that the largest DNA polymerase epsilon subunit POL2A interacts with SU(VAR)3 to 9 homologs SUVH2 and SUVH9. N-SIM (structured illumination microscopy) observation shows that the colocalization of SUVH2 with the meiotic DSB marker γ-H2AX is dependent on POL2A. RNA-seq of male meiocytes demonstrates that POL2A and SUVH2 jointly repress the expression of 865 genes, which have several known characteristics associated with meiotic DSB sites. Bisulfite-seq and small RNA-seq of male meiocytes support the idea that the silencing of these genes by POL2A and SUVH2/9 is likely independent of CHH methylation or 24-nt siRNA accumulation. Moreover, pol2a suvh2 suvh9 triple mutants have more severe defects in meiotic recombination and fertility compared with either pol2a or suvh2 suvh9. Our results not only identify a epigenetic regulatory mechanism for gene silencing in male meiocytes but also reveal roles for DNA polymerase and SUVH2/9 beyond their classic functions in mitosis.

Arabidopsis AAR2, a conserved splicing factor in eukaryotes, acts in microRNA biogenesis.

MicroRNAs (miRNAs) play an essential role in plant growth and development, and as such, their biogenesis is fine-tuned via regulation of the core microprocessor components. Here, we report that Arabidopsis AAR2, a homolog of a U5 snRNP assembly factor in yeast and humans, not only acts in splicing but also promotes miRNA biogenesis. AAR2 interacts with the microprocessor component hyponastic leaves 1 (HYL1) in the cytoplasm, nucleus, and dicing bodies. In aar2 mutants, abundance of nonphosphorylated HYL1, the active form of HYL1, and the number of HYL1-labeled dicing bodies are reduced. Primary miRNA (pri-miRNA) accumulation is compromised despite normal promoter activities of MIR genes in aar2 mutants. RNA decay assays show that the aar2-1 mutation leads to faster degradation of pri-miRNAs in a HYL1-dependent manner, which reveals a previously unknown and negative role of HYL1 in miRNA biogenesis. Taken together, our findings reveal a dual role of AAR2 in miRNA biogenesis and pre-messenger RNA splicing.

SPAAC-NAD-seq, a sensitive and accurate method to profile NAD+-capped transcripts.

Nicotinamide adenine diphosphate (NAD+) is a novel messenger RNA 5' cap in Escherichia coli, yeast, mammals, and Arabidopsis Transcriptome-wide identification of NAD+-capped RNAs (NAD-RNAs) was accomplished through NAD captureSeq, which combines chemoenzymatic RNA enrichment with high-throughput sequencing. NAD-RNAs are enzymatically converted to alkyne-RNAs that are then biotinylated using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Originally applied to E. coli RNA, which lacks the m7G cap, NAD captureSeq was then applied to eukaryotes without extensive verification of its specificity for NAD-RNAs vs. m7G-capped RNAs (m7G-RNAs). In addition, the Cu2+ ion in the CuAAC reaction causes RNA fragmentation, leading to greatly reduced yield and loss of full-length sequence information. We developed an NAD-RNA capture scheme utilizing the copper-free, strain-promoted azide-alkyne cycloaddition reaction (SPAAC). We examined the specificity of CuAAC and SPAAC reactions toward NAD-RNAs and m7G-RNAs and found that both prefer the former, but also act on the latter. We demonstrated that SPAAC-NAD sequencing (SPAAC-NAD-seq), when combined with immunodepletion of m7G-RNAs, enables NAD-RNA identification with accuracy and sensitivity, leading to the discovery of new NAD-RNA profiles in Arabidopsis Furthermore, SPAAC-NAD-seq retained full-length sequence information. Therefore, SPAAC-NAD-seq would enable specific and efficient discovery of NAD-RNAs in prokaryotes and, when combined with m7G-RNA depletion, in eukaryotes.

Parallel analysis of RNA ends reveals global microRNA-mediated target RNA cleavage in maize.

MicroRNAs (miRNAs) are endogenous 20-24-nucleotide non-coding RNAs that play important regulatory roles in many biological processes in eukaryotes. miRNAs modulate the expression of target genes at the post-transcriptional level by transcript cleavage or translational inhibition. The identification of miRNA target genes has been extensively investigated in Arabidopsis and rice, but an in-depth global analysis of miRNA-mediated target regulation is still lacking in maize. Here, we report a transcriptome-wide identification of miRNA targets by analyzing parallel analysis of RNA ends (PARE) datasets derived from nine different tissues at five developmental stages of the maize (Zea mays L.) B73 cultivar. In total, 246 targets corresponding to 60 miRNAs from 25 families were identified, including transcription factors and other genes. In addition, PARE analysis revealed that miRNAs guide specific target transcript cleavage in a tissue-preferential manner. Primary transcripts of MIR159c and MIR169e were found to be cleaved by mature miR159 and miR169, respectively, indicating a negative-feedback regulatory mechanism in miRNA biogenesis. Moreover, several miRNA-target gene pairs involved in seed germination were identified and experimentally validated. Our PARE analyses generated a wide and detailed miRNA-target interaction atlas, which provides a valuable resource for investigating the roles of miRNAs and their targets in maize.

T-LOC: A comprehensive tool to localize and characterize T-DNA integration sites.

Scientists have developed many approaches based on PCR or next-generation sequencing to localize and characterize integrated T-DNAs in transgenic plants generated by Agrobacterium tumefaciens-mediated T-DNA transfer. However, none of these methods has the robust ability to handle all transgenic plants with diversified T-DNA patterns. Utilizing the valuable information in the whole-genome sequencing data of transgenic plants, we have developed a comprehensive approach (T-LOC) to localize and characterize T-DNA integration sites (TISs). We evaluated the performance of T-LOC on genome sequencing data from 48 transgenic rice (Oryza sativa) plants that provide real and unbiased resources of T-DNA integration patterns. T-LOC discovered 75 full TISs and reported a diversified pattern of T-DNA integration: the ideal single-copy T-DNA between two borders, multiple-copy of T-DNAs in tandem or inverted repeats, truncated partial T-DNAs with or without the selection hygromycin gene, the inclusion of T-DNA backbone, the integration at the genome repeat region, and the concatenation of multiple ideal or partial T-DNAs. In addition, we reported that DNA fragments from the two A. tumefaciens plasmids can be fused with T-DNA and integrated into the plant genome. Besides, T-LOC characterizes the genomic changes at TISs, including deletion, duplication, accurate repair, and chromosomal rearrangement. Moreover, we validated the robustness of T-LOC using PCR, Sanger sequencing, and Nanopore sequencing. In summary, T-LOC is a robust approach to studying the TISs independent of the integration pattern and can recover all types of TISs in transgenic plants.

The PROTEIN PHOSPHATASE4 Complex Promotes Transcription and Processing of Primary microRNAs in Arabidopsis.

PROTEIN PHOSPHATASE4 (PP4) is a highly conserved Ser/Thr protein phosphatase found in yeast, plants, and animals. The composition and functions of PP4 in plants are poorly understood. Here, we uncovered the complexity of PP4 composition and function in Arabidopsis (Arabidopsis thaliana) and identified the composition of one form of PP4 containing the regulatory subunit PP4R3A. We show that PP4R3A, together with one of two redundant catalytic subunit genes, PROTEIN PHOSPHATASE X (PPX)1 and PPX2, promotes the biogenesis of microRNAs (miRNAs). PP4R3A is a chromatin-associated protein that interacts with RNA polymerase II and recruits it to the promoters of miRNA-encoding (MIR) genes to promote their transcription. PP4R3A likely also promotes the cotranscriptional processing of miRNA precursors, because it recruits the microprocessor component HYPONASTIC LEAVES1 to MIR genes and to nuclear dicing bodies. Finally, we show that hundreds of introns exhibit splicing defects in pp4r3a mutants. Together, this study reveals roles for Arabidopsis PP4 in transcription and nuclear RNA metabolism.

NAD+-capped RNAs are widespread in the Arabidopsis transcriptome and can probably be translated.

As the most common RNA cap in eukaryotes, the 7-methylguanosine (m7G) cap impacts nearly all processes that a messenger RNA undergoes, such as splicing, polyadenylation, nuclear export, translation, and degradation. The metabolite and redox agent, nicotinamide adenine diphosphate (NAD+), can be used as an initiating nucleotide in RNA synthesis to result in NAD+-capped RNAs. Such RNAs have been identified in bacteria, yeast, and human cells, but it is not known whether they exist in plant transcriptomes. The functions of the NAD+ cap in RNA metabolism or translation are still poorly understood. Here, through NAD captureSeq, we show that NAD+-capped RNAs are widespread in Arabidopsis thaliana NAD+-capped RNAs are predominantly messenger RNAs encoded by the nuclear and mitochondrial genomes, but not the chloroplast genome. NAD+-capped transcripts from the nuclear genome appear to be spliced and polyadenylated. Furthermore, although NAD+-capped transcripts constitute a small proportion of the total transcript pool from any gene, they are enriched in the polysomal fraction and associate with translating ribosomes. Our findings implicate the existence of as yet unknown mechanisms whereby the RNA NAD+ cap interfaces with RNA metabolic processes as well as translation initiation. More importantly, our findings suggest that cellular metabolic and/or redox states may influence, or be regulated by, mRNA NAD+ capping.

Genome-Wide Transcript and Small RNA Profiling Reveals Transcriptomic Responses to Heat Stress.

Because of climate change, crops will experience increasing heat stress. However, the ways in which heat stress affects crop growth and yield at the molecular level remain poorly understood. We generated spatiotemporal mRNA and small RNA transcriptome data, spanning seven tissues at three time points, to investigate the effects of heat stress on vegetative and reproductive development in maize (Zea mays). Among the small RNAs significantly induced by heat stress was a plastid-derived 19-nucleotide small RNA, which is possibly the residual footprint of a pentatricopeptide repeat protein. This suggests that heat stress induces the turnover of certain plastid transcripts. Consistently, genes responsible for photosynthesis in chloroplasts were repressed after heat stress. Analysis also revealed that the abundance of 24-nucletide small interfering RNAs from transposable elements was conspicuously reduced by heat stress in tassels and roots; nearby genes showed a similar expression trend. Finally, specific microRNA and passenger microRNA species were identified, which in other plant species have not before been reported as responsive to heat stress. This study generated an atlas of genome-wide transcriptomic responses to heat stress, revealing several key regulators as potential targets for thermotolerance improvement in maize.

The Arabidopsis MOS4-Associated Complex Promotes MicroRNA Biogenesis and Precursor Messenger RNA Splicing.

In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved, putative RNA helicase MAC7 is a component of the Arabidopsis MAC, and the human MAC7 homolog, Aquarius, is implicated in pre-mRNA splicing. Here, we show that mac7-1, a partial loss-of-function mutant in MAC7, and two other MAC subunit mutants, mac3a mac3b and prl1 prl2 (pleiotropic regulatory locus), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved 11 known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. RNA-seq analysis showed that downregulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants in three subunits of MAC, thus linking MAC function to splicing in Arabidopsis.

Comparative epigenomics reveals evolution of duplicated genes in potato and tomato.

The evolution of duplicated genes after polyploidization has been the subject of many evolutionary biology studies. Potato (Solanum tuberosum) and tomato (Solanum lycopersicum) are the first two sequenced genomes of asterids, and share a common polyploidization event. However, the epigenetic role of DNA methylation on the evolution of duplicated genes derived from polyploidization is not fully understood. Here, we explore the role of the DNA methylation in the evolution of duplicated genes in potato and tomato. The overall levels of DNA methylation are different, although patterns of DNA methylation are similar in potato and tomato. Different types of duplicated genes can display different methylation patterns in potato and tomato. In addition, we found that differences in the methylation levels between duplicated genes were associated with gene expression divergence. In particular, for the majority of duplicated gene pairs, one copy is always hyper- or hypo-methylated compared with the other copy across different tomato fruit ripening stages, and these genes are enriched for specific function related to transcription factor (TF) activity. Furthermore, transcription of hundreds of duplicated TFs was shown to be regulated by DNA methylation during fruit ripening stages in tomato, some of which are well-known fruit ripening TFs. Taken together, our results support the notion that DNA methylation may facilitate divergent evolution of duplicated genes and play roles in important biological processes such as tomato fruit ripening.

DNA methylome analysis provides evidence that the expansion of the tea genome is linked to TE bursts.

DNA methylation is essential for gene regulation, imprinting and silencing of transposable elements (TEs). Although bursts of transposable elements are common in many plant lineages, how plant DNA methylation is related to transposon bursts remains unclear. Here we explore the landscape of DNA methylation of tea, a species thought to have experienced a recent transposon burst event. This species possesses more transposable elements than any other sequenced asterids (potato, tomato, coffee, pepper and tobacco). The overall average DNA methylation levels were found to differ among the tea, potato and tomato genomes, and methylation at CHG sequence sites was found to be significantly higher in tea than that in potato or tomato. Moreover, the abundant TEs resulting from burst events not only resulted in tea developing a very large genome size, but also affected many genes involved in importantly biological processes, including caffeine, theanine and flavonoid metabolic pathway genes. In addition, recently transposed TEs were more heavily methylated than ancient ones, implying that DNA methylation is proportionate to the degree of TE silencing, especially on recent active ones. Taken together, our results show that DNA methylation regulates transposon silencing and may play a role in genome size expansion.

Feedback Regulation of DYT1 by Interactions with Downstream bHLH Factors Promotes DYT1 Nuclear Localization and Anther Development.

Transcriptional regulation is one of the most important mechanisms controlling development and cellular functions in plants and animals. The Arabidopsis thaliana bHLH transcription factor (TF) DYSFUNCTIONL TAPETUM1 (DYT1) is required for normal male fertility and anther development and activates the expression of the bHLH010/bHLH089/bHLH091 genes. Here, we showed that DYT1 is localized to both the cytoplasm and nucleus at anther stage 5 but specifically to the nucleus at anther stage 6 and onward. The bHLH010/bHLH089/bHLH091 proteins have strong nuclear localization signals, interact with DYT1, and facilitate the nuclear localization of DYT1. We further found that the conserved C-terminal BIF domain of DYT1 is required for its dimerization, nuclear localization, transcriptional activation activity, and function in anther development. Interestingly, when the BIF domain of DYT1 was replaced with that of bHLH010, the DYT1(N)-bHLH010(BIF) chimeric protein shows nuclear-preferential localization at anther stage 5 but could not fully rescue the dyt1-3 phenotype, suggesting that the normal spatio-temporal subcellular localization of DYT1 is important for DYT1 function and/or that the BIF domains from different bHLH members might be functionally distinct. Our results support an important positive feedback regulatory mechanism whereby downstream TFs increase the function of an upstream TF by enhancing its nucleus localization through the BIF domain.

Cytological and Transcriptomic Analyses Reveal Important Roles of CLE19 in Pollen Exine Formation.

The CLAVATA3/ESR-RELATED (CLE) peptide signals are required for cell-cell communication in several plant growth and developmental processes. However, little is known regarding the possible functions of the CLEs in the anther. Here, we show that a T-DNA insertional mutant, and dominant-negative (DN) and overexpression (OX) transgenic plants of the CLE19 gene, exhibited significantly reduced anther size and pollen grain number and abnormal pollen wall formation in Arabidopsis (Arabidopsis thaliana). Interestingly, the DN-CLE19 pollen grains showed a more extensively covered surface, but CLE19-OX pollen exine exhibited clearly missing connections in the network and lacked separation between areas that normally form the lacunae. With a combination of cell biological, genetic, and transcriptomic analyses on cle19, DN-CLE19, and CLE19-OX plants, we demonstrated that CLE19-OX plants produced highly vacuolated and swollen aborted microspores (ams)-like tapetal cells, lacked lipidic tapetosomes and elaioplasts, and had abnormal pollen primexine without obvious accumulation of sporopollenin precursors. Moreover, CLE19 is important for the normal expression of more than 1,000 genes, including the transcription factor gene AMS, 280 AMS-downstream genes, and other genes involved in pollen coat and pollen exine formation, lipid metabolism, pollen germination, and hormone metabolism. In addition, the DN-CLE19(+/+) ams(-/-) plants exhibited the ams anther phenotype and ams(+/-) partially suppressed the DN-CLE19 transgene-induced pollen exine defects. These findings demonstrate that the proper amount of CLE19 signal is essential for the normal expression of AMS and its downstream gene networks in the regulation of anther development and pollen exine formation.

MID1 plays an important role in response to drought stress during reproductive development.

Drought during rice reproductive development results in yield loss. It is important to understand the functions of drought-responsive genes in reproductive tissues for improving rice yield under water-deficit conditions. We show here that MID1 (MYB Important for Drought Response1), encoding a putative R-R-type MYB-like transcription factor, can improve rice yield under drought. MID1 was primarily expressed in root and leaf vascular tissues, with low level in the tapetum, and was induced by drought and other abiotic stresses. Compared with wild type, MID1-overexpressing plants were more tolerant to drought at both vegetative and reproductive stages and produced more grains under water stress. MID1-overexpressing plants exhibited less severe anther defects such as deformed anther locules, abnormal tapetum, degenerated microspores and expanded middle layer, with improved pollen fertility and higher seed setting rate. MID1 was localized to the nucleus and could activate gene expression in yeast, and its homologs were identified in many other plants with high levels sequence similarity. In addition, candidate MID1-regulated genes were analyzed using RNA-seq and qRT-PCR, including genes crucial for stress responses and anther development, with altered expressions in the florets of MID1-overexpressing plants and RNAi lines. Furthermore, MID1 could bind to the promoters of two drought-related genes (Hsp17.0 and CYP707A5) and one anther developmental gene (KAR) according to ChIP-qPCR data. Our findings suggest that MID1 is a transcriptional regulator that promotes rice male development under drought by modulating the expressions of drought-related and anther developmental genes and provide valuable information for crop improvement.

APETALA2 antagonizes the transcriptional activity of AGAMOUS in regulating floral stem cells in Arabidopsis thaliana.

APETALA2 (AP2) is best known for its function in the outer two floral whorls, where it specifies the identities of sepals and petals by restricting the expression of AGAMOUS (AG) to the inner two whorls in Arabidopsis thaliana. Here, we describe a role of AP2 in promoting the maintenance of floral stem cell fate, not by repressing AG transcription, but by antagonizing AG activity in the center of the flower. We performed a genetic screen with ag-10 plants, which exhibit a weak floral determinacy defect, and isolated a mutant with a strong floral determinacy defect. This mutant was found to harbor another mutation in AG and was named ag-11. We performed a genetic screen in the ag-11 background to isolate mutations that suppress the floral determinacy defect. Two suppressor mutants were found to harbor mutations in AP2. While AG is known to shut down the expression of the stem cell maintenance gene WUSCHEL (WUS) to terminate floral stem cell fate, AP2 promotes the expression of WUS. AP2 does not repress the transcription of AG in the inner two whorls, but instead counteracts AG activity.

Whole-genome DNA methylation patterns and complex associations with gene structure and expression during flower development in Arabidopsis.

Flower development is a complex process requiring proper spatiotemporal expression of numerous genes. Accumulating evidence indicates that epigenetic mechanisms, including DNA methylation, play essential roles in modulating gene expression. However, few studies have examined the relationship between DNA methylation and floral gene expression on a genomic scale. Here we present detailed analyses of DNA methylomes at single-base resolution for three Arabidopsis floral periods: meristems, early flowers and late flowers. We detected 1.5 million methylcytosines, and estimated the methylation levels for 24 035 genes. We found that many cytosine sites were methylated de novo from the meristem to the early flower stage, and many sites were demethylated from early to late flowers. A comparison of the transcriptome data of the same three periods revealed that the methylation and demethylation processes were correlated with expression changes of >3000 genes, many of which are important for normal flower development. We also found different methylation patterns for three sequence contexts ((m) CG, (m) CHG and (m) CHH) and in different genic regions, potentially with different roles in gene expression.

The DYT1-interacting proteins bHLH010, bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome.

The anther is the male reproductive organ of flowering plants, and the Arabidopsis bHLH transcription factors encoded by DYSFUNCTIONAL TAPETUM1 (DYT1) and ABORTED MICROSPORE (AMS) are required for control of the complex transcriptional networks regulating anther development. Knowledge of the mechanisms by which the bHLH proteins affect this diverse gene expression is quite limited. We examine here three recently duplicated Arabidopsis bHLH genes, bHLH010, bHLH089 and bHLH091, using evolutionary, genetic, morphological and transcriptomic approaches, and uncover their redundant functions in anther development. These three genes are relatively highly expressed in the tapetum of the Arabidopsis anther; single mutants at each of the bHLH010, bHLH089 and bHLH091 loci are developmentally normal, but the various double and triple combinations progressively exhibit increasingly defective anther phenotypes (abnormal tapetum morphology, delayed callose degeneration, and aborted pollen development), indicating their redundant functions in male fertility. Further transcriptomic and molecular analyses suggest that these three proteins act slightly later than DYT1, and also form protein complexes with DYT1, subsequently affecting the correct expression of many DYT1 target genes in the anther development transcriptional network. This study demonstrated that bHLH010, bHLH089 and bHLH091 together are important for the normal transcriptome of the developing Arabidopsis anther, possibly by forming a feed-forward loop with DYT1.

Proteomic and phosphoproteomic analyses reveal extensive phosphorylation of regulatory proteins in developing rice anthers.

Anther development, particularly around the time of meiosis, is extremely crucial for plant sexual reproduction. Meanwhile, cell-to-cell communication between somatic (especial tapetum) cells and meiocytes are important for both somatic anther development and meiosis. To investigate possible molecular mechanisms modulating protein activities during anther development, we applied high-resolution mass spectrometry-based proteomic and phosphoproteomic analyses for developing rice (Oryza sativa) anthers around the time of meiosis (RAM). In total, we identified 4984 proteins and 3203 phosphoproteins with 8973 unique phosphorylation sites (p-sites). Among those detected here, 1544 phosphoproteins are currently absent in the Plant Protein Phosphorylation DataBase (P3 DB), substantially enriching plant phosphorylation information. Mapman enrichment analysis showed that 'DNA repair','transcription regulation' and 'signaling' related proteins were overrepresented in the phosphorylated proteins. Ten genetically identified rice meiotic proteins were detected to be phosphorylated at a total of 25 p-sites; moreover more than 400 meiotically expressed proteins were revealed to be phosphorylated and their phosphorylation sites were precisely assigned. 163 putative secretory proteins, possibly functioning in cell-to-cell communication, are also phosphorylated. Furthermore, we showed that DNA synthesis, RNA splicing and RNA-directed DNA methylation pathways are extensively affected by phosphorylation. In addition, our data support 46 kinase-substrate pairs predicted by the rice Kinase-Protein Interaction Map, with SnRK1 substrates highly enriched. Taken together, our data revealed extensive protein phosphorylation during anther development, suggesting an important post-translational modification affecting protein activity.