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Heat shock transcription factors (HSFs) play a crucial role in heat stress tolerance in vegetative tissues. However, their involvement in reproductive tissues and their post-translational modifications are not well understood. In this study, we identify the E3 ligase XB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (XBAT31) as a key player in the ubiquitination and degradation of HSFB2a/B2b. Our results show that the xbat31 mutant exhibits a higher percentage of unfertile siliques and decreased expression of HSPs in flowers under heat stress conditions compared to the wild type. Conversely, the hsfb2a hsfb2b double mutant displays improved reproductive thermotolerance. We find that XBAT31 interacts with HSFB2a/B2b and mediates their ubiquitination. Furthermore, HSFB2a/B2b ubiquitination is reduced in the xbat31-1 mutant, resulting in higher accumulation of HSFB2a/B2b in flowers under heat stress conditions. Overexpression of HSFB2a or HSFB2b leads to an increase in unfertile siliques under heat stress conditions. Thus, our results dissect the important role of the XBAT31-HSFB2a/B2b module in conferring reproductive thermotolerance in plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Environmental stresses negatively impact reproductive development and yield. Drought stress, in particular, has been examined during Arabidopsis reproductive development at morphological and transcriptomic levels. However, drought-responsive transcriptomic changes at different points in reproductive development remain unclear. Additionally, an investigation of the entire transcriptome at various stages during flower development is of great interest., Results: Here, we treat Arabidopsis plants with well-watered and moderately and severely limiting water amounts when the first flowers reach maturity and generate RNA-seq datasets for early, middle, and late phases during flower development at 5, 6, and 7 days following treatment. Under different drought conditions, flowers in different developmental phases display differential sets of drought-responsive genes (DTGs), including those that are enriched in different GO functional categories, such as transcriptional regulation and response to stresses (early phase), lipid storage (middle phase), and pollen and seed development and metabolic processes (late phase). Some gene families have different members induced at different floral phases, suggesting that similar biochemical functions are carried out by distinct members. Developmentally-regulated genes (DVGs) with differential expression among the three floral phases belong to GO terms that are similar between water conditions, such as development and reproduction, metabolism and transport, and signaling and stress response. However, for different water conditions, such similar GO terms correspond to either distinct gene families or different members of a gene family, suggesting that drought affects the expression of distinct families or family members during reproductive development. A further comparison among transcriptomes of tissues collected on different days after treatment identifies differential gene expression, suggesting age-related genes (ARGs) might reflect the changes in the overall plant physiology in addition to drought response and development., Conclusion: Together, our study provides new insights into global transcriptome reprogramming and candidate genes for drought response, flower development, aging and coordination among these complex biological processes., (© 2024. The Author(s).)

The tapetum, a crucial innermost layer encompassing male reproductive cells within the anther wall, plays a pivotal role in normal pollen development. The transcription factors (TFs) bHLH010/089/091 redundantly facilitate the rapid nuclear accumulation of DYSFUNCTIONAL TAPETUM 1, a gatekeeper TF in the tapetum. Nevertheless, the regulatory mechanisms governing the activity of bHLH010/089/091 remain unknown. In this study, we reveal that caffeoyl coenzyme A O-methyltransferase 1 (CCoAOMT1) is a negative regulator affecting the nuclear localization and function of bHLH010 and bHLH089, probably through their K259 site. Our findings underscore that CCoAOMT1 promotes the nuclear export and degradation of bHLH010 and bHLH089. Intriguingly, elevated CCoAOMT1 expression resulted in defective pollen development, mirroring the phenotype observed in bhlh010 bhlh089 mutants. Moreover, our investigation revealed that the K259A mutation in the bHLH089 protein disrupted its translocation from the nucleus to the cytosol and impeded its degradation induced by CCoAOMT1. Importantly, transgenic plants with the probHLH089::bHLH089 K259A construct failed to rescue proper pollen development or gene expression in bhlh010 bhlh089 mutants. Collectively, these findings emphasize the need to maintain balanced TF homeostasis for male fertility. They firmly establish CCoAOMT1 as a pivotal regulator that is instrumental in achieving equilibrium between the induction of the tapetum transcriptional network and ensuring appropriate anther development., (© 2023. Science China Press.)

Meiotic recombination requires the specific RecA homolog DMC1 recombinase to stabilize strand exchange intermediates in most eukaryotes. Normal DMC1 levels are crucial for its function, yet the regulatory mechanisms of DMC1 stability are unknown in any organism. Here, we show that the degradation of Arabidopsis DMC1 by the 26S proteasome depends on F-box proteins RMF1/2-mediated ubiquitination. Furthermore, RMF1/2 interact with the Skp1 ortholog ASK1 to form the ubiquitin ligase complex SCF RMF1/2 . Genetic analyses demonstrate that RMF1/2, ASK1 and DMC1 act in the same pathway downstream of SPO11-1 dependent meiotic DNA double strand break formation and that the proper removal of DMC1 is crucial for meiotic crossover formation. Moreover, six DMC1 lysine residues were identified as important for its ubiquitination but not its interaction with RMF1/2. Our results reveal mechanistic insights into how the stability of a key meiotic recombinase that is broadly conserved in eukaryotes is regulated., (© 2023. Springer Nature Limited.)

Gametophyte development in angiosperms occurs within diploid sporophytic structures and requires coordinated development; e.g., development of the male gametophyte pollen depends on the surrounding sporophytic tissue, the tapetum. The mechanisms underlying this interaction remain poorly characterized. The peptide CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 19 (CLE19) plays a "braking" role in preventing the harmful overexpression of tapetum transcriptional regulators to ensure normal pollen development in Arabidopsis. However, the CLE19 receptor is unknown. Here, we show that CLE19 interacts directly with the PXY-LIKE1 (PXL1) ectodomain and induces PXL1 phosphorylation. PXL1 is also required for the function of CLE19 in maintaining the tapetal transcriptional regulation of pollen exine genes. Additionally, CLE19 induces the interactions of PXL1 with SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptors required for pollen development. We propose that PXL1 and SERKs act as receptor and coreceptor, respectively, of the extracellular CLE19 signal, thereby regulating tapetum gene expression and pollen development., (© 2023. The Author(s).)

Heterochromatin is essential for genomic integrity and stability in eukaryotes. The mechanisms that regulate meiotic heterochromatin formation remain largely undefined. Here, we show that the catalytic subunit (POL2A) of Arabidopsis DNA polymerase epsilon (POL ε) is required for proper formation of meiotic heterochromatin. The POL2A N terminus interacts with the GHKL adenosine triphosphatase (ATPase) MORC1 (Microrchidia 1), and POL2A is required for MORC1's localization on meiotic heterochromatin. Mutations affecting the POL2A N terminus cause aberrant morphology of meiotic heterochromatin, which is also observed in morc1 . Moreover, the POL2A C-terminal zinc finger domain (ZF1) specifically binds to histone H3.1-H4 dimer or tetramer and is important for meiotic heterochromatin condensation. Interestingly, we also found similar H3.1-binding specificity for the mouse counterpart. Together, our results show that two distinct domains of POL2A, ZF1 and N terminus bind H3.1-H4 and recruit MORC1, respectively, to induce a continuous process of meiotic heterochromatin organization. These activities expand the functional repertoire of POL ε beyond its classic role in DNA replication and appear to be conserved in animals and plants.

Organogenesis in plants occurs across all stages of the life cycle. Although previous studies have identified many genes as important for either vegetative or reproductive development at the RNA level, global information on translational and post-translational levels remains limited. In this study, six Arabidopsis stages/organs were analyzed using quantitative proteomics and phosphoproteomics, identifying 2187 non-redundant proteins and evidence for 1194 phosphoproteins. Compared to the expression observed in cauline leaves, the expression of 1445, 1644, and 1377 proteins showed greater than 1.5-fold alterations in stage 1-9 flowers, stage 10-12 flowers, and open flowers, respectively. Among these, 294 phosphoproteins with 472 phosphorylation sites were newly uncovered, including 275 phosphoproteins showing differential expression patterns, providing molecular markers and possible candidates for functional studies. Proteins encoded by genes preferentially expressed in anther (15), meiocyte (4), or pollen (15) were enriched in reproductive organs, and mutants of two anther-preferentially expressed proteins, acos5 and mee48 , showed obviously reduced male fertility with abnormally organized pollen exine. In addition, more phosphorylated proteins were identified in reproductive stages (1149) than in the vegetative organs (995). The floral organ-preferential phosphorylation of GRP17, CDC2/CDKA.1, and ATSK11 was confirmed with western blot analysis. Moreover, phosphorylation levels of CDPK6 and MAPK6 and their interacting proteins were elevated in reproductive tissues. Overall, our study yielded extensive data on protein expression and phosphorylation at six stages/organs and provides an important resource for future studies investigating the regulatory mechanisms governing plant development.

Cohesin, a multisubunit protein complex, is required for holding sister chromatids together during mitosis and meiosis. The recruitment of cohesin by the sister chromatid cohesion 2/4 (SCC2/4) complex has been extensively studied in Saccharomyces cerevisiae mitosis, but its role in mitosis and meiosis remains poorly understood in multicellular organisms, because complete loss-of-function of either gene causes embryonic lethality. Here, we identified a weak allele of Atscc2 (Atscc2-5) that has only minor defects in vegetative development but exhibits a significant reduction in fertility. Cytological analyses of Atscc2-5 reveal multiple meiotic phenotypes including defects in chromosomal axis formation, meiosis-specific cohesin loading, homolog pairing and synapsis, and AtSPO11-1-dependent double strand break repair. Surprisingly, even though AtSCC2 interacts with AtSCC4 in vitro and in vivo, meiosis-specific knockdown of AtSCC4 expression does not cause any meiotic defect, suggesting that the SCC2-SCC4 complex has divergent roles in mitosis and meiosis. SCC2 homologs from land plants have a unique plant homeodomain (PHD) motif not found in other species. We show that the AtSCC2 PHD domain can bind to the N terminus of histones and is required for meiosis but not mitosis. Taken together, our results provide evidence that unlike SCC2 in other organisms, SCC2 requires a functional PHD domain during meiosis in land plants., Competing Interests: The authors have declared that no competing interests exist.

Backgroud: Many Gram-negative bacteria use N-acyl-homoserine lactones (AHLs) to communicate each other and to coordinate their collective behaviors. Recently, accumulating evidence shows that host plants are able to sense and respond to bacterial AHLs. Once primed, plants are in an altered state that enables plant cells to more quickly and/or strongly respond to subsequent pathogen infection or abiotic stress., Results: In this study, we report that pretreatment with N-3-oxo-octanoyl-homoserine lactone (3OC8-HSL) confers resistance against the pathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (PstDC3000) in Arabidopsis. Pretreatment with 3OC8-HSL and subsequent pathogen invasion triggered an augmented burst of hydrogen peroxide, salicylic acid accumulation, and fortified expression of the pathogenesis-related genes PR1 and PR5. Upon PstDC3000 challenge, plants treated with 3OC8-HSL showed increased activities of defense-related enzymes including peroxidase, catalase, phenylalanine ammonialyase, and superoxide dismutase. In addition, the 3OC8-HSL-primed resistance to PstDC3000 in wild-type plants was impaired in plants expressing the bacterial NahG gene and in the npr1 mutant. Moreover, the expression levels of isochorismate synthases (ICS1), a critical salicylic acid biosynthesis enzyme, and two regulators of its expression, SARD1 and CBP60g, were potentiated by 3OC8-HSL pretreatment followed by pathogen inoculation., Conclusions: Our data indicate that 3OC8-HSL primes the Arabidopsis defense response upon hemibiotrophic bacterial infection and that 3OC8-HSL-primed resistance is dependent on the SA signaling pathway. These findings may help establish a novel strategy for the control of plant disease.

Meiosis is a critical process for sexual reproduction. During meiosis, genetic information on homologous chromosomes is shuffled through meiotic recombination to produce gametes with novel allelic combinations. Meiosis and recombination are orchestrated by several mechanisms including regulation by small RNAs (sRNAs). Our previous work in Arabidopsis ( Arabidopsis thaliana ) meiocytes showed that meiocyte-specific sRNAs (ms-sRNAs) have distinct characteristics, including positive association with the coding region of genes that are transcriptionally upregulated during meiosis. Here, we characterized the ms-sRNAs in two important crops, soybean ( Glycine max ) and cucumber ( Cucumis sativus ). Ms-sRNAs in soybean have the same features as those in Arabidopsis, suggesting that they may play a conserved role in eudicots. We also investigated the profiles of microRNAs (miRNAs) and phased secondary small interfering RNAs in the meiocytes of all three species. Two conserved miRNAs, miR390 and miR167, are highly abundant in the meiocytes of all three species. In addition, we identified three novel cucumber miRNAs. Intriguingly, our data show that the previously identified phased secondary small interfering RNA pathway involving soybean-specific miR4392 is more abundant in meiocytes. These results showcase the conservation and divergence of ms-sRNAs in flowering plants, and broaden our understanding of sRNA function in crop species., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Faithful chromosome segregation is required for both mitotic and meiotic cell divisions and is regulated by multiple mechanisms including the anaphase-promoting complex/cyclosome (APC/C), which is the largest known E3 ubiquitin-ligase complex and has been implicated in regulating chromosome segregation in both mitosis and meiosis in animals. However, the role of the APC/C during plant meiosis remains largely unknown. Here, we show that Arabidopsis APC8 is required for male meiosis. We used a combination of genetic analyses, cytology and immunolocalisation to define the function of AtAPC8 in male meiosis. Meiocytes from apc8-1 plants exhibit several meiotic defects including improper alignment of bivalents at metaphase I, unequal chromosome segregation during anaphase II, and subsequent formation of polyads. Immunolocalisation using an antitubulin antibody showed that APC8 is required for normal spindle morphology. We also observed mitotic defects in apc8-1, including abnormal sister chromatid segregation and microtubule morphology. Our results demonstrate that Arabidopsis APC/C is required for meiotic chromosome segregation and that APC/C-mediated regulation of meiotic chromosome segregation is a conserved mechanism among eukaryotes., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Meiotic recombination contributes to the maintenance of the association between homologous chromosomes (homologs) and ensures the accurate segregation of homologs during anaphase I, thus facilitating the redistribution of alleles among progeny. Meiotic recombination is initiated by the programmed formation of DNA double strand breaks, the repair of which requires DNA synthesis, but the role of DNA synthesis proteins during meiosis is largely unknown. Here, we hypothesized that the lagging strand-specific DNA Polymerase δ (POL δ) might be required for meiotic recombination, based on a previous analysis of DNA Replication Factor1 that suggested a role for lagging strand synthesis in meiotic recombination. In Arabidopsis ( Arabidopsis thaliana ), complete mutation of the catalytic subunit of POL δ, encoded by AtPOLD1 , leads to embryo lethality. Therefore, we used a meiocyte-specific knockdown strategy to test this hypothesis. Reduced expression of AtPOLD1 in meiocytes caused decreased fertility and meiotic defects, including incomplete synapsis, the formation of multivalents, chromosome fragmentation, and improper segregation. Analysis of meiotic crossover (CO) frequencies showed that AtPOLD1 RNAi plants had significantly fewer interference-sensitive COs than the wild type, indicating that AtPOL δ participates in type I CO formation. AtPOLD1 RNAi atpol2a double mutant meiocytes displayed more severe meiotic phenotypes than those of either single mutant, suggesting that the function of AtPOLD1 and AtPOL2A is not identical in meiotic recombination. Given that POL δ is highly conserved among eukaryotes, we hypothesize that the described role of POL δ here in meiotic recombination likely exists widely in eukaryotes., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis ( Arabidopsis thaliana ) and meiocytes of spo11 - 1 , a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1 - LIKE1 and RAD51 ), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination., (© 2019 American Society of Plant Biologists. All rights reserved.)

For most eukaryotes, sexual reproduction is a fundamental process that requires meiosis. In turn, meiosis typically depends on a reciprocal exchange of DNA between each pair of homologous chromosomes, known as a crossover (CO), to ensure proper chromosome segregation. The frequency and distribution of COs are regulated by intrinsic and extrinsic environmental factors, but much more is known about the molecular mechanisms governing the former compared to the latter. Here we show that elevated temperature induces meiotic hyper-recombination in Arabidopsis thaliana and we use genetic analysis with mutants in different recombination pathways to demonstrate that the extra COs are derived from the major Type I interference sensitive pathway. We also show that heat-induced COs are not the result of an increase in DNA double-strand breaks and that the hyper-recombinant phenotype is likely specific to thermal stress rather than a more generalized stress response. Taken together, these findings provide initial mechanistic insight into how environmental cues modulate plant meiotic recombination and may also offer practical applications., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Root organogenesis involves cell division, differentiation and expansion. The molecular mechanisms regulating root development are not fully understood. In this study, we identified poly(adenosine diphosphate (ADP)-ribose) polymerases (PARPs) as new players in root development. PARP catalyzes poly(ADP-ribosyl)ation of proteins by repeatedly adding ADP-ribose units onto proteins using nicotinamide adenine dinucleotide (NAD + ) as the donor. We found that inhibition of PARP activities by 3-aminobenzomide (3-AB) increased the growth rates of both primary and lateral roots, leading to a more developed root system. The double mutant of Arabidopsis PARPs, parp1parp2, showed more rapid primary and lateral root growth. Cyclin genes regulating G1-to-S and G2-to-M transition were up-regulated upon treatment by 3-AB. The proportion of 2C cells increased while cells with higher DNA ploidy declined in the roots of treated plants, resulting in an enlarged root meristematic zone. The expression level of PARP2 was very low in the meristematic zone but high in the maturation zone, consistent with a role of PARP in inhibiting mitosis and promoting cell differentiation. Our results suggest that PARPs play an important role in root development by negatively regulating root cell division., (© 2017 Institute of Botany, Chinese Academy of Sciences.)

Meiotic recombination is required for proper homologous chromosome segregation in plants and other eukaryotes. The eukaryotic RAD51 gene family has seven ancient paralogs with important roles in mitotic and meiotic recombination. Mutations in mammalian RAD51 homologs RAD51C and XRCC3 lead to embryonic lethality. In the model plant Arabidopsis thaliana, RAD51C and XRCC3 homologs are not essential for vegetative development but are each required for somatic and meiotic recombination, but the mechanism of RAD51C and XRCC3 in meiotic recombination is unclear. The non-lethal Arabidopsis rad51c and xrcc3 null mutants provide an opportunity to study their meiotic functions. Here, we show that AtRAD51C and AtXRCC3 are components of the RAD51-dependent meiotic recombination pathway and required for normal AtRAD51 localization on meiotic chromosomes. In addition, AtRAD51C interacts with both AtRAD51 and AtXRCC3 in vitro and in vivo, suggesting that these proteins form a complex (es). Comparison of AtRAD51 foci in meiocytes from atrad51, atrad51c, and atxrcc3 single, double and triple heterozygous mutants further supports an interaction between AtRAD51C and AtXRCC3 that enhances AtRAD51 localization. Moreover, atrad51c-/+ atxrcc3-/+ double and atrad51-/+ atrad51c-/+ atxrcc3-/+ triple heterozygous mutants have defects in meiotic recombination, suggesting the role of the AtRAD51C-AtXRCC3 complex in meiotic recombination is in part AtRAD51-dependent. Together, our results support a model in which direct interactions between the RAD51C-XRCC3 complex and RAD51 facilitate RAD51 localization on meiotic chromosomes and RAD51-dependent meiotic recombination. Finally, we hypothesize that maintenance of RAD51 function facilitated by the RAD51C-XRCC3 complex could be highly conserved in eukaryotes.

High temperatures have a great impact on plant reproductive development and subsequent fruit and seed set, but the underlying molecular mechanisms are not well understood. We used transcriptome profiling to investigate the effect of heat stress on reproductive development of Arabidopsis thaliana plants and observed distinct response patterns in vegetative versus reproductive tissues. Exposure to heat stress affected reproductive developmental programs, including early phases of anther/ovule development and meiosis. Also, genes participating in the unfolded protein response (UPR) were enriched in the reproductive tissue-specific genes that were upregulated by heat. Moreover, we found that the UPR-deficient bzip28 bzip60 double mutant was sensitive to heat stresses and had reduced silique length and fertility. Comparison of heat-responsive wild type versus bzip28 bzip60 plants identified 521 genes that were regulated by bZIP28 and bZIP60 upon heat stress during reproductive stages, most of which were noncanonical UPR genes. Chromatin immunoprecipitation coupled with high-throughput sequencing analyses revealed 133 likely direct targets of bZIP28 in Arabidopsis seedlings subjected to heat stress, including 27 genes that were also upregulated by heat during reproductive development. Our results provide important insights into heat responsiveness in Arabidopsis reproductive tissues and demonstrate the protective roles of the UPR for maintaining fertility upon heat stress., (© 2017 American Society of Plant Biologists. All rights reserved.)

Hundreds of leucine-rich repeat receptor-like kinases (LRR-RLKs) play indispensable roles in a wide range of plant developmental and physiological processes. The mechanisms controlling LRR-RLKs at a basal and inactive status are essential but rarely studied. BKI1 is the only reported inhibitor of receptor kinases in Arabidopsis, which negatively regulates BRI1 in the brassinosteroid pathway. In this study, we found that BKI1 can also interact with another important LRR-RLK, ERECTA (ER). Phenotypic analysis showed that BKI1 and ER together regulate plant architecture, including pedicel orientation, which is a newly reported phenotype in the BR- and ER-mediated developmental processes. Gene expression analysis revealed that BKI1 regulates a subset of ER-responsive genes. Kinase assays demonstrated that BKI1 inhibits ER kinase activity. In addition, the release of BKI1 inhibition on ER signaling relies largely on BRI1 activation. Our data provide significant insights into the regulation and activation of RLKs and suggest that BKI1 functions as a common suppressor of the BRI1 and ER signaling pathways., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

The C-repeat binding factor (CBF) is crucial for regulation of cold response in higher plants. In Arabidopsis, the mechanism of CBF3-caused growth retardation is still unclear. Our present work shows that CBF3 shares the similar repression of bioactive gibberellin (GA) as well as upregulation of DELLA proteins with CBF1 and -2. Genetic analysis reveals that DELLAs play an essential role in growth reduction mediated by CBF1, -2, -3 genes. The in vivo and in vitro evidences demonstrate that GA2-oxidase 7 gene is a novel CBF3 regulon. Meanwhile, DELLAs contribute to cold induction of CBF1, -2, -3 genes through interaction with jasmonate (JA) signaling. We conclude that CBF3 promotes DELLAs accumulation through repressing GA biosynthesis and DELLAs positively regulate CBF3 involving JA signaling. CBFs and DELLAs collaborate to retard plant growth in response to low temperature.

N-acyl-homoserine lactones (AHL) are the quorum-sensing (QS) signal molecules used by many gram-negative bacteria to coordinate their collective behavior in a population. Recent evidence demonstrates their roles in plant root growth and defense responses. AtMYB44 is a multifaceted transcriptional factor that functions in many physiological processes in plants but whether AtMYB44 modulates the plant response to AHL with aspects of primary root elongation remains unknown. Here, we show that the expression of AtMYB44 was upregulated upon treatment with N-3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). The stimulatory effect of 3OC6-HSL on primary root elongation was abolished in the AtMYB44 functional-deficiency mutant atmby44. In contrast, an enhanced promoting-impact of 3OC6-HSL on primary root growth was observed in AtMYB44-overexpressing plant MYB44OTA. Cellular analysis indicated that the prolonged primary root elicited by 3OC6-HSL is the consequence of increased cell division in the meristem zone and enhanced cell elongation in the elongation zone, and AtMYB44 may act as a positive regulator in this process. Furthermore, we demonstrated that AtMYB44 might participate in the 3OC6-HSL-mediated primary root growth via regulating the expression of cytokinin- and auxin-related genes. The data establish a genetic connection between the regulatory role of AtMYB44 in phytohormones-related gene expression and plant response to the bacterial QS signal.

As the male reproductive organ of flowering plants, the stamen consists of the anther and filament. Previous studies on stamen development mainly focused on single gene functions by genetic methods or gene expression changes using comparative transcriptomic approaches, especially in model plants such as Arabidopsis thaliana However, studies on Arabidopsis anther protein expression and post-translational modifications are still lacking. Here we report proteomic and phosphoproteomic studies on developing Arabidopsis anthers at stages 4-7 and 8-12. We identified 3908 high-confidence phosphorylation sites corresponding to 1637 phosphoproteins. Among the 1637 phosphoproteins, 493 were newly identified, with 952 phosphorylation sites. Phosphopeptide enrichment prior to LC-MS analysis facilitated the identification of low-abundance proteins and regulatory proteins, thereby increasing the coverage of proteomic analysis, and facilitated the analysis of more regulatory proteins. Thirty-nine serine and six threonine phosphorylation motifs were uncovered from the anther phosphoproteome and further analysis supports that phosphorylation of casein kinase II, mitogen-activated protein kinases, and 14-3-3 proteins is a key regulatory mechanism in anther development. Phosphorylated residues were preferentially located in variable protein regions among family members, but they were they were conserved across angiosperms in general. Moreover, phosphorylation might reduce activity of reactive oxygen species scavenging enzymes and hamper brassinosteroid signaling in early anther development. Most of the novel phosphoproteins showed tissue-specific expression in the anther according to previous microarray data. This study provides a community resource with information on the abundance and phosphorylation status of thousands of proteins in developing anthers, contributing to understanding post-translational regulatory mechanisms during anther development., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Chromosome condensation, a process mediated by the condensin complex, is essential for proper chromosome segregation during cell division. Unlike rapid mitotic chromosome condensation, meiotic chromosome condensation occurs over a relatively long prophase I and is unusually complex due to the coordination with chromosome axis formation and homolog interaction. The molecular mechanisms that regulate meiotic chromosome condensation progression from prophase I to metaphase I are unclear. Here, we show that the Arabidopsis thaliana meiotic PHD-finger protein MMD1/DUET is required for progressive compaction of prophase I chromosomes to metaphase I bivalents. The MMD1 PHD domain is required for its function in chromosome condensation and binds to methylated histone tails. Transcriptome analysis and qRT-PCR showed that several condensin genes exhibit significantly reduced expression in mmd1 meiocytes. Furthermore, MMD1 specifically binds to the promoter region of the condensin subunit gene CAP-D3 to enhance its expression. Moreover, cap-d3 mutants exhibit similar chromosome condensation defects, revealing an MMD1-dependent mechanism for regulating meiotic chromosome condensation, which functions in part by promoting condensin gene expression. Together, these discoveries provide strong evidence that the histone reader MMD1/DUET defines an important step for regulating the progression of meiotic prophase I chromosome condensation., (© 2016 American Society of Plant Biologists. All rights reserved.)

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., (© 2016 American Society of Plant Biologists. All rights reserved.)

Background: The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown., Results: Here we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type, but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development., Conclusions: Our results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.

Cell division requires proper spindle assembly; a surveillance pathway, the spindle assembly checkpoint (SAC), monitors whether the spindle is normal and correctly attached to kinetochores. The SAC proteins regulate mitotic chromosome segregation by affecting CDC20 (Cell Division Cycle 20) function. However, it is unclear whether CDC20 regulates meiotic spindle assembly and proper homolog segregation. Here, we show that the Arabidopsis thaliana CDC20.1 gene is indispensable for meiosis and male fertility. We demonstrate that cdc20.1 meiotic chromosomes align asynchronously and segregate unequally and the metaphase I spindle has aberrant morphology. Comparison of the distribution of meiotic stages at different time points between the wild type and cdc20.1 reveals a delay of meiotic progression from diakinesis to anaphase I. Furthermore, cdc20.1 meiocytes exhibit an abnormal distribution of a histone H3 phosphorylation mark mediated by the Aurora kinase, providing evidence that CDC20.1 regulates Aurora localization for meiotic chromosome segregation. Further evidence that CDC20.1 and Aurora are functionally related was provided by meiosis-specific knockdown of At-Aurora1 expression, resulting in meiotic chromosome segregation defects similar to those of cdc20.1. Taken together, these results suggest a critical role for CDC20.1 in SAC-dependent meiotic chromosome segregation., (© 2015 American Society of Plant Biologists. All rights reserved.)

Poly(ADP-ribosyl)ation is a reversible post-translational modification of proteins, characterized by the addition of poly(ADP-ribose) (PAR) to proteins by poly(ADP-ribose) polymerase (PARP), and removal of PAR by poly(ADP-ribose) glycohydrolase (PARG). Three PARPs and two PARGs have been found in Arabidopsis, but their respective roles are not fully understood. In this study, the functions of each PARP and PARG in DNA repair were analyzed based on their mutant phenotypes under genotoxic stresses. Double or triple mutant analysis revealed that PARP1 and PARP2, but not PARP3, play a similar but not critical role in DNA repair in Arabidopsis seedlings. PARG1 and PARG2 play an essential and a minor role, respectively under the same conditions. Mutation of PARG1 results in increased DNA damage level and enhanced cell death in plants after bleomycin treatment. PARG1 expression is induced primarily in root and shoot meristems by bleomycin and induction of PARG1 is dependent on ATM and ATR kinases. PARG1 also antagonistically modulates the DNA repair process by preventing the over-induction of DNA repair genes. Our study determined the contribution of each PARP and PARG member in DNA repair and indicated that PARG1 plays a critical role in this process.

Plants flower in an appropriate season to allow sufficient vegetative development and position flower development in favorable environments. In Arabidopsis, CONSTANS (CO) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) promote flowering by inducing FLOWER LOCUS T (FT) expression in the long-day afternoon. The CO protein is present in the morning but could not activate FT expression due to unknown negative mechanisms, which prevent premature flowering before the day length reaches a threshold. Here, we report that TARGET OF EAT1 (TOE1) and related proteins interact with the activation domain of CO and CO-like (COL) proteins and inhibit CO activity. TOE1 binds to the FT promoter near the CO-binding site, and reducing TOE function results in a morning peak of the FT mRNA. In addition, TOE1 interacts with the LOV domain of FKF1 and likely interferes with the FKF1-CO interaction, resulting in partial degradation of the CO protein in the afternoon to prevent premature flowering., (© 2015 Zhang et al.; Published by Cold Spring Harbor Laboratory Press.)

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., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.

Background: Drought is a major constraint that leads to extensive losses to agricultural yield worldwide. The potential yield is largely determined during inflorescence development. However, to date, most investigations on plant response to drought have focused on vegetative development. This study describes the morphological changes of reproductive development and the comparison of transcriptomes under various drought conditions., Results: The plants grown were studied under two drought conditions: minimum for successful reproduction (45-50% soil water content, moderate drought, MD) and for survival (30-35%, severe drought, SD). MD plants can produce similar number of siliques on the main stem and similar number of seeds per silique comparing with well-water plants. The situation of SD plants was much worse than MD plants. The transcriptomes of inflorescences were further investigated at molecular level using microarrays. Our results showed more than four thousands genes with differential expression under severe drought and less than two thousand changed under moderate drought condition (with 2-fold change and q-value < 0.01). We found a group of genes with increased expression as the drought became more severe, suggesting putative adaptation to the dehydration. Interestingly, we also identified genes with alteration only under the moderate but not the severe drought condition, indicating the existence of distinct sets of genes responsive to different levels of water availability. Further cis-element analyses of the putative regulatory sequences provided more information about the underlying mechanisms for reproductive responses to drought, suggesting possible novel candidate genes that protect those developing flowers under drought stress., Conclusions: Different pathways may be activated in response to moderate and severe drought in reproductive tissues, potentially helping plant to maximize its yield and balance the resource consumption between vegetative and reproductive development under dehydration stresses.

During angiosperm microsporogenesis, callose serves as a temporary wall to separate microsporocytes and newly formed microspores in the tetrad. Abnormal callose deposition and dissolution can lead to degeneration of developing microspores. However, genes and their regulation in callose metabolism during microsporogenesis still remain largely unclear. Here, we demonstrated that the Arabidopsis (Arabidopsis thaliana) CALLOSE DEFECTIVE MICROSPORE1 (CDM1) gene, encoding a tandem CCCH-type zinc finger protein, plays an important role in regulation of callose metabolism in male meiocytes and in integrity of newly formed microspores. First, quantitative reverse transcription PCR and in situ hybridization analyses showed that the CDM1 gene was highly expressed in meiocytes and the tapetum from anther stages 4 to 7. In addition, a transfer DNA insertional cdm1 mutant was completely male sterile. Moreover, light microscopy of anther sections revealed that microspores in the mutant anther were initiated, and then degenerated soon afterward with callose deposition defects, eventually leading to male sterility. Furthermore, transmission electron microscopy demonstrated that pollen exine formation was severely affected in the cdm1 mutant. Finally, we found that the cdm1 mutation affected the expression of callose synthesis genes (CALLOSE SYNTHASE5 and CALLOSE SYNTHASE12) and potential callase-related genes (A6 and MYB80), as well as three other putative β-1,3-glucanase genes. Therefore, we propose that the CDM1 gene regulates callose metabolism during microsporogenesis, thereby promoting Arabidopsis male fertility.

The eukaryotic RAD51 gene family has seven ancient paralogs conserved between plants and animals. Among these, RAD51, DMC1, RAD51C and XRCC3 are important for homologous recombination and/or DNA repair, whereas single mutants in RAD51B, RAD51D or XRCC2 show normal meiosis, and the lineages they represent diverged from each other evolutionarily later than the other four paralogs, suggesting possible functional redundancy. The function of Arabidopsis RAD51B, RAD51D and XRCC2 genes in mitotic DNA repair and meiosis was analyzed using molecular genetic, cytological and transcriptomic approaches. The relevant double and triple mutants displayed normal vegetative and reproductive growth. However, the triple mutant showed greater sensitivity than single or double mutants to DNA damage by bleomycin. RNA-Seq transcriptome analysis supported the idea that the triple mutant showed DNA damage similar to that caused by bleomycin. On bleomycin treatment, many genes were altered in the wild-type but not in the triple mutant, suggesting that the RAD51 paralogs have roles in the regulation of gene transcription, providing an explanation for the hypersensitive phenotype of the triple mutant to bleomycin. Our results provide strong evidence that Arabidopsis XRCC2, RAD51B and RAD51D have complex functions in somatic DNA repair and gene regulation, arguing for further studies of these ancient genes that have been maintained in both plants and animals during their long evolutionary history., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Drought dramatically affects plant growth and crop yield, but previous studies primarily examined responses to drought during vegetative development. Here, to study responses to drought during reproductive development, we grew Arabidopsis thaliana plants with limited water, under conditions that allowed the plants to initiate and complete reproduction. Drought treatment from just after the onset of flowering to seed maturation caused an early arrest of floral development and sterility. After acclimation, plants showed reduced fertility that persisted throughout reproductive development. Floral defects included abnormal anther development, lower pollen viability, reduced filament elongation, ovule abortion, and failure of flowers to open. Drought also caused differential expression of 4153 genes, including flowering time genes flowering locus t, suppressor of overexpression of CO1, and leafy, genes regulating anther and pistil development, and stress-related transcription factors. Mutant phenotypes of hypersensitivity to drought and fewer differentially expressed genes suggest that dehydration response element B1A may have an important function in drought response in flowers. A more severe filament elongation defect under drought in myb21 plants demonstrated that appropriate stamen development requires MYB domain protein 21 under drought conditions. Our study reveals a regulatory cascade in reproductive responses and acclimation under drought.

The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors (RopGEFs) and in turn may be activated by an unknown factor through releasing RopGEF1's auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprk1,2,5 triple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 (DN-PRK2) inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1(90-457), implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 (RopGEF1S460A and RopGEF1S480A) eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regulator of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.

Several genes encoding transcription factors have been shown to be essential for male fertility in plants, suggesting that transcriptional regulation is a major mechanism controlling anther development in Arabidopsis. DYSFUNCTIONAL TAPETUM 1 (DYT1), a putative bHLH transcription factor, plays a critical role in regulating tapetum function and pollen development. Here, we compare the transcriptomes of young anthers of wild-type and the dyt1 mutant, demonstrating that DYT1 is upstream of at least 22 genes encoding transcription factors and regulates the expression of a large number of genes, including genes involved in specific metabolic pathways. We also show that DYT1 can bind to DNA in a sequence-specific manner in vitro, and induction of DYT1 activity in vivo activated the expression of the downstream transcription factor genes MYB35 and MS1. We generated DYT1-SRDX transgenic plants whose fertility was dramatically reduced, implying that DYT1 probably acts as a transcriptional activator. Furthermore, we used yeast two-hybrid assays to show that DYT1 forms homodimers and heterodimers with other bHLH transcription factors. Our results demonstrate the important role of DYT1 in regulating anther transcriptome and function, and supporting normal pollen development., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

DNA double-strand break (DSB) formation is the initial event for meiotic recombination catalyzed by the conserved Spo11 protein. In Arabidopsis, several proteins have been reported to be involved in DSB formation. Here, we report an Arabidopsis DSB forming (DFO) gene in Arabidopsis that is involved in DSB formation. The dfo mutant exhibits reduced fertility, producing polyads with an abnormal number of microspores, unlike the tetrads in the wild type. The dfo meiocytes were defective in homologous chromosome synapsis and segregation. Genetic analysis revealed that the homologous recombination of Atdfo-1 is severely affected in meiotic prophase I. DFO encodes a protein without any known conserved domain. There was no homologue identified outside the plant kingdom, indicating that AtDFO is a plant-specific protein. AtMRE11 has been reported to be responsible for processing SPO11-generated DSBs. The Atmre11 mutant displays chromosome fragmentation during meiosis. However, the Atdfo Atmre11 double mutant had no such chromosome fragmentation, indicating that AtDFO is required for DSB formation., (© 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.)

Meiotic recombination, including crossovers (COs) and gene conversions (GCs), impacts natural variation and is an important evolutionary force. COs increase genetic diversity by redistributing existing variation, whereas GCs can alter allelic frequency. Here, we sequenced Arabidopsis Landsberg erecta (Ler) and two sets of all four meiotic products from a Columbia (Col)/Ler hybrid to investigate genome-wide variation and meiotic recombination at nucleotide resolution. Comparing Ler and Col sequences uncovered 349,171 Single Nucleotide Polymorphisms (SNPs), 58,085 small and 2315 large insertions/deletions (indels), with highly correlated genome-wide distributions of SNPs, and small indels. A total of 443 genes have at least 10 nonsynonymous substitutions in protein-coding regions, with enrichment for disease-resistance genes. Another 316 genes are affected by large indels, including 130 genes with complete deletion of coding regions in Ler. Using the Arabidopsis qrt1 mutant, two sets of four meiotic products were generated and analyzed by sequencing for meiotic recombination, representing the first tetrad analysis with whole-genome sequencing in a nonfungal species. We detected 18 COs, six of which had an associated GC event, and four GCs without COs (NCOs), and revealed that Arabidopsis GCs are likely fewer and with shorter tracts than those in yeast. Meiotic recombination and chromosome assortment events dramatically redistributed genome variation in meiotic products, contributing to population diversity. In particular, meiosis provides a rapid mechanism to generate copy-number variation (CNV) of sequences that have different chromosomal positions in Col and Ler.

Background: In flowering plants, the development of male reproductive organs is controlled precisely to achieve successful fertilization and reproduction. Despite the increasing knowledge of genes that contribute to anther development, the regulatory mechanisms controlling this process are still unclear., Results: In this study, we analyzed the transcriptome profiles of early anthers of sterile mutants aborted microspores (ams) and found that 1,368 genes were differentially expressed in ams compared to wild type anthers, affecting metabolism, transportation, ubiquitination and stress response. Moreover, the lack of significant enrichment of potential AMS binding sites (E-box) in the promoters of differentially expressed genes suggests both direct and indirect regulation for AMS-dependent regulation of anther transcriptome involving other transcription factors. Combining ams transcriptome profiles with those of two other sterile mutants, spl/nzz and ems1/exs, expression of 3,058 genes were altered in at least one mutant. Our investigation of expression patterns of major transcription factor families, such as bHLH, MYB and MADS, suggested that some closely related homologs of known anther developmental genes might also have similar functions. Additionally, comparison of expression levels of genes in different organs suggested that anther-preferential genes could play important roles in anther development., Conclusion: Analysis of ams anther transcriptome and its comparison with those of spl/nzz and ems1/exs anthers uncovered overlapping and distinct sets of regulated genes, including those encoding transcription factors and other proteins. These results support an expanded regulatory network for early anther development, providing a series of hypotheses for future experimentation.

During meiotic recombination, induced double-strand breaks (DSBs) are processed into crossovers (COs) and non-COs (NCO); the former are required for proper chromosome segregation and fertility. DNA synthesis is essential in current models of meiotic recombination pathways and includes only leading strand DNA synthesis, but few genes crucial for DNA synthesis have been tested genetically for their functions in meiosis. Furthermore, lagging strand synthesis has been assumed to be unnecessary. Here we show that the Arabidopsis thaliana DNA replication factor C1 (RFC1) important for lagging strand synthesis is necessary for fertility, meiotic bivalent formation, and homolog segregation. Loss of meiotic RFC1 function caused abnormal meiotic chromosome association and other cytological defects; genetic analyses with other meiotic mutations indicate that RFC1 acts in the MSH4-dependent interference-sensitive pathway for CO formation. In a rfc1 mutant, residual pollen viability is MUS81-dependent and COs exhibit essentially no interference, indicating that these COs form via the MUS81-dependent interference-insensitive pathway. We hypothesize that lagging strand DNA synthesis is important for the formation of double Holliday junctions, but not alternative recombination intermediates. That RFC1 is found in divergent eukaryotes suggests a previously unrecognized and highly conserved role for DNA synthesis in discriminating between recombination pathways., Competing Interests: The authors have declared that no competing interests exist.

Meiosis is essential for eukaryotic sexual reproduction, with two consecutive rounds of nuclear divisions, allowing production of haploid gametes. Information regarding the meiotic transcriptome should provide valuable clues about global expression patterns and detailed gene activities. Here we used RNA sequencing to explore the transcriptome of a single plant cell type, the Arabidopsis male meiocyte, detecting the expression of approximately 20 000 genes. Transcription of introns of >400 genes was observed, suggesting previously unannotated exons. More than 800 genes may be preferentially expressed in meiocytes, including known meiotic genes. Of the 3378 Pfam gene families in the Arabidopsis genome, 3265 matched meiocyte-expressed genes, and 18 gene families were over-represented in male meiocytes, including transcription factor and other regulatory gene families. Expression was detected for many genes thought to encode meiosis-related proteins, including MutS homologs (MSHs), kinesins and ATPases. We identified more than 1000 orthologous gene clusters that are also expressed in meiotic cells of mouse and fission yeast, including 503 single-copy genes across the three organisms, with a greater number of gene clusters shared between Arabidopsis and mouse than either share with yeast. Interestingly, approximately 5% transposable element genes were apparently transcribed in male meiocytes, with a positive correlation to the transcription of neighboring genes. In summary, our RNA-Seq transcriptome data provide an overview of gene expression in male meiocytes and invaluable information for future functional studies., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)

Plant reproductive development is a complex process with diploid and haploid phases, including male and female organogenesis, meiosis, gametogenesis, pollination and fertilization. A number of regulatory mechanisms control both diploid and haploid cell division and differentiation, especially cell-cell signaling pathways mediated by receptor-linked protein kinases with prominent roles in early male development, and hormonal signaling pathways crucial for later events in male and female reproductive development. Furthermore, transcriptional networks control the proper formation of specific cell layers and embryo sac cell specification., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The plant-specific GRAS/SCL transcription factors play diverse roles in plant development and stress responses. In this study, a poplar SCL gene, PeSCL7, was functionally characterized in Arabidopsis thaliana, especially with regard to its role in abiotic stress resistance. Expression analysis in poplar revealed that PeSCL7 was induced by drought and high salt stresses, but was repressed by gibberellic acid (GA) treatment in leaves. Transient expression of GFP-PeSCL7 in onion epidermal cells revealed that the PeSCL7 protein was localized in the nucleus. Transgenic Arabidopsis plants overexpressing PeSCL7 showed enhanced tolerance to drought and salt treatments. The activity of two stress-responsive enzymes was increased in transgenic seedlings. Taken together, these results suggest that PeSCL7 encodes a member of the stress-responsive GRAS/SCL transcription factors that is potentially useful for engineering drought- and salt-tolerant trees.

The development of anther and pollen is important for male reproduction, and this process is coordinately regulated by many external and internal cues. In this study, we systematically examined the male reproductive phenotypes of a series of brassinosteroid biosynthetic and signaling mutants and found that, besides the expected cell-expansion defects, these mutants also showed reduced pollen number, viability, and release efficiency. These defects were related with abnormal tapetum and microspore development. Using both real-time quantitative RT-PCR and microarray experiments, we found that the expression of many key genes required for anther and pollen development was suppressed in these mutants. ChIP analysis demonstrated that BES1, an important transcription factor for brassinosteroid signaling, could directly bind to the promoter regions of genes encoding transcription factors essential for anther and pollen development, SPL/NZZ, TDF1, AMS, MS1, and MS2. Taken together, these data lead us to propose that brassinosteroids control male fertility at least in part via directly regulating key genes for anther and pollen development in Arabidopsis. Our work provides a unique mechanism to explain how a phytohormone regulates an essential genetic program for plant development.

Background: Although the overwhelming majority of genes found in angiosperms are members of gene families, and both gene- and genome-duplication are pervasive forces in plant genomes, some genes are sufficiently distinct from all other genes in a genome that they can be operationally defined as 'single copy'. Using the gene clustering algorithm MCL-tribe, we have identified a set of 959 single copy genes that are shared single copy genes in the genomes of Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa. To characterize these genes, we have performed a number of analyses examining GO annotations, coding sequence length, number of exons, number of domains, presence in distant lineages, such as Selaginella and Physcomitrella, and phylogenetic analysis to estimate copy number in other seed plants and to demonstrate their phylogenetic utility. We then provide examples of how these genes may be used in phylogenetic analyses to reconstruct organismal history, both by using extant coverage in EST databases for seed plants and de novo amplification via RT-PCR in the family Brassicaceae., Results: There are 959 single copy nuclear genes shared in Arabidopsis, Populus, Vitis and Oryza ["APVO SSC genes"]. The majority of these genes are also present in the Selaginella and Physcomitrella genomes. Public EST sets for 197 species suggest that most of these genes are present across a diverse collection of seed plants, and appear to exist as single or very low copy genes, though exceptions are seen in recently polyploid taxa and in lineages where there is significant evidence for a shared large-scale duplication event. Genes encoding proteins localized in organelles are more commonly single copy than expected by chance, but the evolutionary forces responsible for this bias are unknown.Regardless of the evolutionary mechanisms responsible for the large number of shared single copy genes in diverse flowering plant lineages, these genes are valuable for phylogenetic and comparative analyses. Eighteen of the APVO SSC single copy genes were amplified in the Brassicaceae using RT-PCR and directly sequenced. Alignments of these sequences provide improved resolution of Brassicaceae phylogeny compared to recent studies using plastid and ITS sequences. An analysis of sequences from 13 APVO SSC genes from 69 species of seed plants, derived mainly from public EST databases, yielded a phylogeny that was largely congruent with prior hypotheses based on multiple plastid sequences. Whereas single gene phylogenies that rely on EST sequences have limited bootstrap support as the result of limited sequence information, concatenated alignments result in phylogenetic trees with strong bootstrap support for already established relationships. Overall, these single copy nuclear genes are promising markers for phylogenetics, and contain a greater proportion of phylogenetically-informative sites than commonly used protein-coding sequences from the plastid or mitochondrial genomes., Conclusions: Putatively orthologous, shared single copy nuclear genes provide a vast source of new evidence for plant phylogenetics, genome mapping, and other applications, as well as a substantial class of genes for which functional characterization is needed. Preliminary evidence indicates that many of the shared single copy nuclear genes identified in this study may be well suited as markers for addressing phylogenetic hypotheses at a variety of taxonomic levels.

Normal flower development requires the termination of stem cell activities in the floral meristem. The floral regulator AGAMOUS (AG) is necessary for this termination and represses the expression of the stem cell determinant WUSCHEL (WUS), but the repression mechanism was not clear. A recent study by Sun and colleagues (pp. 1791-1804) in this issue of Genes & Development has identified a direct target of AG, KNUCKLES (KNU), which encodes a transcriptional repressor of WUS, providing a key missing link in floral meristem determinacy.

The proteome of the Arabidopsis flower has not been extensively studied previously. Here, we report a proteomic analysis of the wild type Arabidopsis flower. Using both two-dimensional electrophoresis/mass spectrometry (2-DGE/MS) and multi-dimensional protein identification technology (MudPIT) approaches, we identified 2,446 proteins. Although a single experiment or analysis uncovered only a subset of the proteins we identified, a combination of multiple experiments and analyses facilitated the detection of a greater number of proteins. When proteins are grouped according to RNA expression levels revealed by microarray experiments, we found that proteins encoded by genes with relatively high levels of expression were detected with greater frequencies. On the other hand, at the level of the individual gene/protein, there was not a good correlation between protein spot intensity and microarray values. We also obtained strong evidence for post-translational modification from 2-DGE and MudPIT data. We detected proteins that are annotated to function in protein synthesis, folding, modification, and degradation, as well as the presence of regulatory proteins such as transcription factors and protein kinases. Finally, sequence and evolutionary analysis of genes for active methyl group metabolisms suggests that these genes are highly conserved. Our results allow the formulation of hypotheses regarding post-translational regulation of proteins in the flower, providing new understanding about Arabidopsis flower development and physiology.

Mitogen-activated protein kinase (MAPK) and leucine-rich repeat receptor-like kinase (LRR-RLK) signaling pathways have been shown to regulate diverse aspects of plant growth and development. In Arabidopsis, proper anther development relies on intercellular communication to coordinate cell proliferation and differentiation. Two closely related genes encoding MAPKs, MPK3 and MPK6, function redundantly in regulating stomatal patterning. Although the mpk6 mutant has reduced fertility, the function of MPK3 and MPK6 in anther development has not been characterized. Similarly, the ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 genes encoding LRR-RLKs function together to direct stomatal cell fate specification and the er-105 erl1-2 erl2-1 triple mutant is sterile. Because the mpk3 mpk6 double null mutant is embryo lethal, anther development was characterized in the viable mpk3/+ mpk6/- and er-105 erl1-2 erl2-1 mutants. We found that both mutant anthers usually fail to form one or more of the four anther lobes, with the er-105 erl1-2 erl2-1 triple mutant exhibiting more severe phenotypes than those of the mpk3/+ mpk6/- mutant. The somatic cell layers of the differentiated mutant lobes appeared larger and more disorganized than that of wild-type. In addition, the er-105 erl1-2 erl2-1 triple mutant has a reduced number of stamens, the majority of which possess completely undifferentiated or under-differentiated anthers. Furthermore, sometimes, the mpk3/+ mpk6/- mutant anthers do not dehisce, and the er-105 erl1-2 erl2-1 anthers were not observed to dehisce. Therefore, our results indicate that both ER/ERL1/ERL2 and MPK3/MPK6 play important roles in normal anther lobe formation and anther cell differentiation. The close functional relationship between these genes in other developmental processes and the similarities in anther developmental phenotypes of the two types of mutants reported here further suggest the possibility that these genes might also function in the same pathway to regulate anther cell division and differentiation.

Gene duplication is important for gene family evolution, allowing for functional divergence and innovation. In flowering plants, duplicated genes are widely observed, and functional redundancy of closely related duplicates has been reported, but few cases of functional divergence of close duplicates have been described. Here, we show that the Arabidopsis AtKIN14a and AtKIN14b genes encoding highly similar kinesins are two of the most closely related Arabidopsis paralogs, which were formed by a duplication event that occurred after the split of Arabidopsis and poplar. In addition, AtKIN14a and AtKIN14b exhibit varying degrees of coding sequence divergence. Further genetic studies of plants carrying atkin14a and/or atkin14b mutations indicate that, although these two genes have similar functions, there is clear evidence for functional divergence. Although both genes are important for male and female meiosis, AtKIN14a plays a more critical role in male meiosis than AtKIN14b. Moreover, either one of these two genes is necessary and sufficient for gametophyte development, indicating that they are redundant for this function. Therefore, AtKIN14a and AtKIN14b together play important roles in controlling plant reproductive development. Our results suggest that the AtKIN14a and AtKIN14b genes have retained similar functions in gametophyte development and female meiosis, but have evolved partially distinct functions in male meiosis, with AtKIN14a playing a more substantive role.

Boundary formation is crucial for organ development in multicellular eukaryotes. In higher plants, boundaries that separate the organ primordia from their surroundings have relatively low rates of cell proliferation. This cellular feature is regulated by the actions of certain boundary-specifying genes, whose ectopic expression in organs can cause inhibition of organ growth. Here, we show that the Arabidopsis thaliana ASYMMETRIC LEAVES1 and 2 (AS1 and AS2) and JAGGED (JAG) genes function in the sepal and petal primordia to repress boundary-specifying genes for normal development of the organs. Loss-of-function as1 jag and as2 jag double mutants produced extremely tiny sepals and petals. Analysis of a cell-cycle marker HISTONE4 revealed that cell division in sepal primordia of the double mutant was inhibited. Moreover, these abnormal sepals and petals exhibited ectopic overexpression of the boundary-specifying genes PETAL LOSS (PTL) and CUP-SHAPED COTYLEDON1 [corrected] and 2 (CUC1 and CUC2). Loss of PTL or CUC1 and CUC2 functions in the as1 jag background could partially rescue the tiny sepal and petal phenotypes, supporting the model that the tiny sepal/petal phenotypes are caused, at least in part, by ectopic expression of boundary-specifying genes. Together, our data reveal a previously unrecognized fundamental regulation by which AS1, AS2, and JAG act to define sepal and petal from their boundaries.