Auxin production in diploid microsporocytes is necessary and sufficient for early stages of pollen development.

Gametophytic development in Arabidopsis depends on nutrients and cell wall materials from sporophytic cells. However, it is not clear whether hormones and signaling molecules from sporophytic tissues are also required for gametophytic development. Herein, we show that auxin produced by the flavin monooxygenases YUC2 and YUC6 in the sporophytic microsporocytes is essential for early stages of pollen development. The first asymmetric mitotic division (PMI) of haploid microspores is the earliest event in male gametophyte development. Microspore development in yuc2yuc6 double mutants arrests before PMI and consequently yuc2yuc6 fail to produce viable pollens. Our genetic analyses reveal that YUC2 and YUC6 act as sporophytic genes for pollen formation. We further show that ectopic production of auxin in tapetum, which provides nutrients for pollen development, fails to rescue the sterile phenotypes of yuc2yuc6. In contrast, production of auxin in either microsporocytes or microspores rescued the defects of pollen development in yuc2yuc6 double mutants. Our results demonstrate that local auxin biosynthesis in sporophytic microsporocytic cells and microspore controls male gametophyte development during the generation transition from sporophyte to male gametophyte.

An Essential Role for miRNA167 in Maternal Control of Embryonic and Seed Development.

Maternal cells play a critical role in ensuring the normal development of embryos, endosperms, and seeds. Mutations that disrupt the maternal control of embryogenesis and seed development are difficult to identify. Here, we completely deleted four MICRORNA167 (MIR167) genes in Arabidopsis (Arabidopsis thaliana) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion of MIR167A phenocopied plants overexpressing miRNA167-resistant versions of Auxin Response Factor6 (ARF6) or ARF8, two miRNA167 targets. Both the mir167a mutant and the ARF overexpression lines were defective in anther dehiscence and ovule development. Serendipitously, we found that the mir167a (♀) × wild type (♂) crosses failed to produce normal embryos and endosperms, despite the findings that embryos with either mir167a+/- or mir167a-/- genotypes developed normally when mir167a+/- plants were self-pollinated, revealing a central role of MIR167A in maternal control of seed development. The mir167a phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternal control. Moreover, we found that mir167a mutants flowered significantly later than wild-type plants, a phenotype that was not observed in the ARF overexpression lines. We show that the reproductive defects of mir167a mutants were suppressed by a decrease of activities of ARF6, ARF8, or both. Our results clearly demonstrate that MIR167A is the predominant MIR167 member in regulating Arabidopsis reproduction and that MIR167A acts as a maternal gene that functions largely through ARF6 and ARF8.

The Regulation of Sporopollenin Biosynthesis Genes for Rapid Pollen Wall Formation.

Sporopollenin is the major component of the outer pollen wall (sexine). It is synthesized using a pathway of approximately eight genes in Arabidopsis (Arabidopsis thaliana). MALE STERILITY188 (MS188) and its direct upstream regulator ABORTED MICROSPORES (AMS) are two transcription factors essential for tapetum development. Here, we show that all the sporopollenin biosynthesis proteins are specifically expressed in the tapetum and are secreted into anther locules. MS188, a MYB transcription factor expressed in the tapetum, directly regulates the expression of POLYKETIDE SYNTHASE A (PKSA), PKSB, MALE STERILE2 (MS2), and a CYTOCHROME P450 gene (CYP703A2). By contrast, the expression of CYP704B1, ACYL-COA SYNTHETASE5 (ACOS5), TETRAKETIDE a-PYRONE REDUCTASE1 (TKPR1) and TKPR2 are significantly reduced in ams mutants but not affected in ms188 mutants. However, MS188 but not AMS can activate the expression of CYP704B1, ACOS5, and TKPR1 In ms188, dominant suppression of MS188 homologs reduced the expression of these genes, suggesting that MS188 and other MYB family members play redundant roles in activating their expression. The expression of some sporopollenin synthesis genes (PKSA, PKSB, TKPR2, CYP704B1, and ACOS5) was rescued when MS188 was expressed in ams Therefore, MS188 is a key regulator for activation of sporopollenin synthesis, and AMS and MS188 may form a feed-forward loop that activates the expression of the sporopollenin biosynthesis pathway for rapid pollen wall formation.

jaw-1D: a gain-of-function mutation responsive to paramutation-like induction of epigenetic silencing.

The Arabidopsis thaliana gain-of-function T-DNA insertion mutant jaw-1D produces miR319A, a microRNA that represses genes encoding CIN-like TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), a family of transcription factors that play key roles in leaf morphogenesis. In this study, we show that jaw-1D is responsive to paramutation-like epigenetic silencing. A genetic cross of jaw-1D with the polycomb gene mutant curly leaf-29 (clf-29) leads to attenuation of the jaw-1D mutant plant phenotype. This induced mutation, jaw-1D*, was associated with down-regulation of miR319A, was heritable independently from clf-29, and displayed paramutation-like non-Mendelian inheritance. Down-regulation of miR319A in jaw-1D* was linked to elevated levels of histone H3 lysine 9 dimethylation and DNA methylation at the CaMV35S enhancer located within the activation-tagging T-DNA of the jaw-1D locus. Examination of 21 independent T-DNA insertion mutant lines revealed that 11 could attenuate the jaw-1D mutant phenotype in a similar way to the paramutation induced by clf-29. These paramutagenic mutant lines shared the common feature that their T-DNA insertion was present as multi-copy tandem repeats and contained high levels of CG and CHG methylation. Our results provide important insights into paramutation-like epigenetic silencing, and caution against the use of jaw-1D in genetic interaction studies.

SDG2-Mediated H3K4me3 Is Crucial for Chromatin Condensation and Mitotic Division during Male Gametogenesis in Arabidopsis.

Epigenetic reprogramming occurring during reproduction is crucial for both animal and plant development. Histone H3 Lys 4 trimethylation (H3K4me3) is an evolutionarily conserved epigenetic mark of transcriptional active euchromatin. While much has been learned in somatic cells, H3K4me3 deposition and function in gametophyte is poorly studied. Here, we demonstrate that SET DOMAIN GROUP2 (SDG2)-mediated H3K4me3 deposition participates in epigenetic reprogramming during Arabidopsis male gametogenesis. We show that loss of SDG2 barely affects meiosis and cell fate establishment of haploid cells. However, we found that SDG2 is critical for postmeiotic microspore development. Mitotic cell division progression is partly impaired in the loss-of-function sdg2-1 mutant, particularly at the second mitosis setting up the two sperm cells. We demonstrate that SDG2 is involved in promoting chromatin decondensation in the pollen vegetative nucleus, likely through its role in H3K4me3 deposition, which prevents ectopic heterochromatic H3K9me2 speckle formation. Moreover, we found that derepression of the LTR retrotransposon ATLANTYS1 is compromised in the vegetative cell of the sdg2-1 mutant pollen. Consistent with chromatin condensation and compromised transcription activity, pollen germination and pollen tube elongation, representing the key function of the vegetative cell in transporting sperm cells during fertilization, are inhibited in the sdg2-1 mutant. Taken together, we conclude that SDG2-mediated H3K4me3 is an essential epigenetic mark of the gametophyte chromatin landscape, playing critical roles in gamete mitotic cell cycle progression and pollen vegetative cell function during male gametogenesis and beyond.

Fine regulation of ARF17 for anther development and pollen formation.

BACKGROUND: In Arabidopsis, the tapetum and microsporocytes are critical for pollen formation. Previous studies have shown that ARF17 is expressed in microsporocytes and tetrads and directly regulates tetrad wall synthesis for pollen formation. ARF17 is the direct target of miR160, and promoterARF17::5mARF17 (5mARF17/WT) transgenic plants, which have five silent mutations within the miR160-complementary domain, are sterile. RESULTS: Here, we found that ARF17 is also expressed in the tapetum, which was defective in arf17 mutants. Compared with arf17 mutants, 5mARF17/WT plants had abnormal tapetal cells and tetrads but were less vacuolated in the tapetum. Immunocytochemical assays showed that the ARF17 protein over-accumulated in tapetum, microsporocytes and tetrads of 5mARF17/WT plants at early anther stages, but its expression pattern was not affected during anther development. 5mARF17 driven by its native promoter did not rescue the arf17 male-sterile phenotype. The expression of 5mARF17 driven by the tapetum-specific promoter A9 led to a defective tapetum and male sterility in transgenic plants. These results suggest that the overexpression of ARF17 in the tapetum and microsporocytes of 5mARF17/WT plants leads to male sterility. Microarray data revealed that an abundance of genes involved in transcription and translation are ectopically expressed in 5mARF17/WT plants. CONCLUSIONS: Our work shows that ARF17 plays an essential role in anther development and pollen formation, and ARF17 expression under miR160 regulation is critical for its function during anther development.

Ethylene Activates the EIN2-EIN3/EIL1 Signaling Pathway in Tapetum and Disturbs Anther Development in Arabidopsis.

Ethylene was previously reported to repress stamen development in both cucumber and Arabidopsis. Here, we performed a detailed analysis of the effect of ethylene on anther development. After ethylene treatment, stamens but not pistils display obvious developmental defects which lead to sterility. Both tapetum and microspores (or microsporocytes) degenerated after ethylene treatment. In ein2-1 and ein3-1 eil1-1 mutants, ethylene treatment did not affect their fertility, indicating the effects of ethylene on anther development are mediated by EIN2 and EIN3/EIL1 in vivo. The transcription of EIN2 and EIN3 are activated by ethylene in the tapetum layer. However, ectopic expression of EIN3 in tapetum did not induce significant anther defects, implying that the expression of EIN3 are regulated post transcriptional level. Consistently, ethylene treatment induced the accumulation of EIN3 in the tapetal cells. Thus, ethylene not only activates the transcription of EIN2 and EIN3, but also stabilizes of EIN3 in the tapetum to disturb its development. The expression of several ethylene related genes was significantly increased, and the expression of the five key transcription factors required for tapetum development was decreased after ethylene treatment. Our results thus point out that ethylene inhibits anther development through the EIN2-EIN3/EIL1 signaling pathway. The activation of this signaling pathway in anther wall, especially in the tapetum, induces the degeneration of the tapetum and leads to pollen abortion.

Characterization of the AE7 gene in Arabidopsis suggests that normal cell proliferation is essential for leaf polarity establishment.

The formation of leaf polarity is critical for leaf morphogenesis. In this study, we characterized and cloned an Arabidopsis gene, AS1/2 ENHANCER7 (AE7), which is required for both leaf adaxial-abaxial polarity formation and normal cell proliferation. The ae7 mutant exhibited leaf adaxial-abaxial polarity defects and double mutants combining ae7 with the leaf polarity mutants as1 (asymmetric leaves1), as2, rdr6 (RNA-dependent RNA polymerase6) or ago7/zip (argonaute7/zippy) all resulted in plants with an apparently enhanced loss of adaxial leaf identity. In addition, ae7 also showed decreased cell proliferation in both leaves and roots, compensated by increased cell sizes in leaves. AE7 encodes a protein conserved in many eukaryotic organisms, ranging from unicellular yeasts to humans; however, the functions of AE7 family members from other species have not been reported. In situ hybridization revealed that AE7 is expressed in a spotted pattern in plant tissues, similar to cell-cycle marker genes such as HISTONE4. Moreover, the ae7 endoploidy and expression analysis of several cell-cycle marker genes in ae7 suggest that the AE7 gene is required for cell cycle progression. As the previously characterized 26S proteasome and ribosome mutants also affect both leaf adaxial-abaxial polarity and cell proliferation, similar to the defects in ae7, we propose that normal cell proliferation may be essential for leaf polarity establishment. Possible models for how cell proliferation influences leaf adaxial-abaxial polarity establishment are discussed.

In Situ Detection of Mature miRNAs in Plants Using LNA-Modified DNA Probes.

MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions for both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression has been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs, because the mature miRNAs are too short (~21-nucleotides) to exhibit stable hybridization signals. In recent years, the Locked nucleic acid (LNA) modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.

The ARGONAUTE10 gene modulates shoot apical meristem maintenance and establishment of leaf polarity by repressing miR165/166 in Arabidopsis.

The shoot apical meristem (SAM) of angiosperms comprises a group of undifferentiated cells which divide to maintain the meristem and also give rise to all the above-ground structures of the plant. Previous studies revealed that the Arabidopsis ARGONAUTE10 [AGO10, also called PINHEAD (PNH) or ZWILLE (ZLL)] gene is one of the critical SAM regulators, but the mechanism by which AGO10 modulates the SAM is unknown. In the present study we show that AGO10 genetically represses microRNA165/166 (miR165/166) for SAM maintenance as well as establishment of leaf adaxial-abaxial polarity. Levels of miR165/166 in leaves and embryonic SAMs of pnh/zll/ago10 mutants are abnormally elevated, leading to a reduction in the quantity of homeodomain-leucine zipper (HD-ZIP) III gene transcripts, the targets of miR165/166. This reduction is the primary cause of pnh/zll SAM and leaf defects, because the aberrant pnh/zll phenotypes were partially rescued by either increasing levels of HD-ZIP III transcripts or decreasing levels of miR165/166 in the SAM and leaf. Furthermore, plants with an abnormal apex were more frequent among pnh/zll rdr6 and pnh/zll ago7 double mutants and increased levels of miR165/166 were detected in rdr6 apices. These results indicate that AGO10 and RDR6/AGO7 may act in parallel in modulating accumulation of miR165/166 for normal plant development.

Slowing development restores the fertility of thermo-sensitive male-sterile plant lines.

Temperature-sensitive genic male sterility (TGMS) lines are widely used in the breeding of hybrid crops1,2, but by what means temperature as a general environmental factor reverses the fertility of different TGMS lines remains unknown. Here, we identified an Arabidopsis TGMS line named reversible male sterile (rvms) that is fertile at low temperature (17 °C) and encodes a GDSL lipase. Cytological observations and statistical analysis showed that low temperature slows pollen development. Further screening of restorers of rvms, as well as crossing with a slow-growth line at normal temperature (24 °C), demonstrate that slowing of development overcomes the defects of rvms microspores and allows them to develop into functional pollen. Several other Arabidopsis TGMS lines were identified, and their fertility was also restored by slowing of development. Given that male reproductive development is conserved3, we propose that slowing of development is a general mechanism applicable to the sterility-fertility conversion of TGMS lines from different plant species.

The Canonical E2Fs Are Required for Germline Development in Arabidopsis.

A number of cell fate determinations, including cell division, cell differentiation, and programmed cell death, intensely occur during plant germline development. How these cell fate determinations are regulated remains largely unclear. The transcription factor E2F is a core cell cycle regulator. Here we show that the Arabidopsis canonical E2Fs, including E2Fa, E2Fb, and E2Fc, play a redundant role in plant germline development. The e2fa e2fb e2fc (e2fabc) triple mutant is sterile, although its vegetative development appears normal. On the one hand, the e2fabc microspores undergo cell death during pollen mitosis. Microspores start to die at the bicellular stage. By the tricellular stage, the majority of the e2fabc microspores are degenerated. On the other hand, a wild type ovule often has one megaspore mother cell (MMC), whereas the majority of e2fabc ovules have two to three MMCs. The subsequent female gametogenesis of e2fabc mutant is aborted and the vacuole is severely impaired in the embryo sac. Analysis of transmission efficiency showed that the canonical E2Fs from both male and female gametophyte are essential for plant gametogenesis. Our study reveals that the canonical E2Fs are required for plant germline development, especially the pollen mitosis and the archesporial cell (AC)-MMC transition.

SDG2-mediated H3K4 methylation is required for proper Arabidopsis root growth and development.

Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several Arabidopsis TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the sdg2 mutants, SDG2 function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the sdg2 mutant root phenotype and demonstrate that SDG2 is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the sdg2 mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that SDG2 and CHROMATIN ASSEMBLY FACTOR-1 act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.

In situ detection of mature miRNAs in plants using LNA-modified DNA probes.

MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions of both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression have been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains the entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs because the mature miRNAs are too short (~21 nt) to exhibit stable hybridization signals. In recent years, the locked nucleic acid (LNA)-modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant -tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.

Two types of cis-acting elements control the abaxial epidermis-specific transcription of the MIR165a and MIR166a genes.

During leaf development, polarity formation is critical for leaf morphogenesis and functions. This process is regulated by several components including two microRNAs, miR165 and 166, which negatively regulate transcription factor genes PHABULOSA, PHAVOLUTA and REVOLUTA. Although miR165 and 166 are known to be accumulated in the abaxial leaf domain, how this pattern is determined is largely unknown. Here we report that the MIR165a and 166a genes are predominantly transcribed in the abaxial epidermis, and this transcript distribution pattern is controlled by two types of cis-acting elements. Our results suggest a model for the polar accumulation of MIR165 and 166 transcripts.