RiceAntherNet: a gene co‐expression network for identifying anther and pollen development genes

In plants, normal anther and pollen development involves many important biological events and complex molecular regulatory coordination. Understanding gene regulatory relationships during male reproductive development is essential for fundamental biology and crop breeding. In this work, we developed a rice gene co‐expression network for anther development (RiceAntherNet) that allows prediction of gene regulatory relationships during pollen development. RiceAntherNet was generated from 57 rice anther tissue microarrays across all developmental stages. The microarray datasets from nine rice male sterile mutants, including msp1‐4, ostdl1a, gamyb‐2, tip2, udt1‐1, tdr, eat1‐1, ptc1 and mads3‐4, were used to explore and test the network. Among the changed genes, three clades showing differential expression patterns were constructed to identify genes associated with pollen formation. Many of these have known roles in pollen development, for example, seven genes in Clade 1 (OsABCG15, OsLAP5, OsLAP6, DPW, CYP703A3, OsNP1 and OsCP1) are involved in rice pollen wall formation. Furthermore, Clade 1 contained 12 genes whose predicted orthologs in Arabidopsis have been reported as key during pollen development and may play similar roles in rice. Genes in Clade 2 are expressed earlier than Clade 1 (anther stages 2–9), while genes in Clade 3 are expressed later (stages 10–12). RiceAntherNet serves as a valuable tool for identifying novel genes during plant anther and pollen development. A website is provided ( https://www.cpib.ac.uk/anther/riceindex.html ) to present the expression profiles for gene characterization. This will assist in determining the key relationships between genes, thus enabling characterization of critical genes associated with anther and pollen regulatory networks.     

The unusual gynoecium structure and extragynoecial pollen-tube pathway in <Emphasis Type="Italic">Phytolacca</Emphasis> (Phytolaccaceae)

The fusion of carpels into a unified compound gynoecium is considered a dominant feature of angiosperm evolution and it also occurs by postgenital fusion during the gynoecium development in some apocarpous species. However, we found the reverse process, the separation of carpels from combined carpel primordia, during the development of the gynoecium in Phytolacca. Semithin sectioning and scanning electron microscopy were utilised to observe the structure and development of the gynoecia in Phytolacca acinosa and Phytolacca americana, fluorescence microscopy was utilised to observe the pollen tube growth in the gynoecia of the two species, and the topology method was applied to analyze the relationship between the gynoecium structure and pollen tube pathway. Although the gynoecia of P. acinosa and P. americana are both syncarpous, the degree of carpel fusion in the mature gynoecia of the two syncarpous species is different as a result of variant developmental processes. However, change in the degree of carpel fusion during the development of gynoecia in Phytolacca does not affect pollen tube growth because of the existence of the extragynoecial pollen-tube pathway. Thus, the change in the degree of carpel fusion in Phytolacca is primarily the result of diversification of developmental processes related to selection pressure.     

Control of Reproductive Floral Organ Identity Specification in Arabidopsis by the C Function Regulator AGAMOUS

The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.     

Inception of maleness: auxin contribution to flower masculinization in the dioecious cactus Opuntia stenopetala

In Opuntia stenopetala, flowers initiate as hermaphrodite; however, at maturity, only the stamens in male flowers and the gynoecium in female flowers become functional. At early developmental stages, growth and morphogenesis of the gynoecium in male flowers cease, forming a short style lacking stigmatic tissue at maturity. Here, an analysis of the masculinization process of this species and its relationship with auxin metabolism during gynoecium morphogenesis is presented. Histological analysis and scanning electron microscopy were performed; auxin levels were immunoanalyzed and exogenous auxin was applied to developing gynoecia. Male flower style-tissue patterning revealed morphological defects in the vascular bundles, stylar canal, and transmitting tissue. These features are similar to those observed in Arabidopsis thaliana mutant plants affected in auxin transport, metabolism, or signaling. Notably, when comparing auxin levels between male and female gynoecia from O. stenopetala at an early developmental stage, we found that they were particularly low in the male gynoecium. Consequently, exogenous auxin application on male gynoecia partially restored the defects of gynoecium development. We therefore hypothesize that, the arrest in male flower gynoecia patterning could be related to altered auxin homeostasis; alternatively, the addition of auxin could compensate for the lack of another unknown factor affecting male flower gynoecium development.     

Imprinting of the Polycomb Group Gene MEDEA Serves as a Ploidy Sensor in Arabidopsis

Balanced maternal and paternal genome contributions are a requirement for successful seed development. Unbalanced contributions often cause seed abortion, a phenomenon that has been termed “triploid block.” Misregulation of imprinted regulatory genes has been proposed to be the underlying cause for abnormalities in growth and structure of the endosperm in seeds with deviating parental contributions. We identified a mutant forming unreduced pollen that enabled us to investigate direct effects of unbalanced parental genome contributions on seed development and to reveal the underlying molecular mechanism of dosage sensitivity. We provide evidence that parent-of-origin–specific expression of the Polycomb group (PcG) gene MEDEA is causally responsible for seed developmental aberrations in Arabidopsis seeds with increased paternal genome contributions. We propose that imprinted expression of PcG genes is an evolutionary conserved mechanism to balance parental genome contributions in embryo nourishing tissues.     

Fluorescence-tagged transgenic lines reveal genetic defects in pollen growth--application to the eIF3 complex

Background

Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.

Methodology/Principal Findings

Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation.

Conclusion/Significance

eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.     

A Role for Arabidopsis PUCHI in Floral Meristem Identity and Bract Suppression

At the onset of flowering, the Arabidopsis thaliana primary inflorescence meristem starts to produce flower meristems on its flank. Determination of floral fate is associated with changes in the growth pattern and expression of meristem identity genes and suppression of a subtending leaf called a bract. Here, we show a role in floral fate determination and bract suppression for the PUCHI gene, an AP2/EREBP family gene that has previously been reported to play roles in lateral root morphogenesis. Mutations in PUCHI cause partial conversion of flowers to inflorescences, indicating that PUCHI is required for flower meristem identity. PUCHI is transiently expressed in the early flower meristem and accelerates meristem bulging while it prevents the growth of the bract primordium. The function of PUCHI in floral fate determination and bract suppression overlaps that of the BLADE-ON-PETIOLE1 (BOP1) and BOP2 genes, which encode a pair of redundant regulatory proteins involved in various developmental processes, including leaf morphogenesis and flower patterning. We also show that PUCHI acts together with BOP1 and BOP2 to promote expression of LEAFY and APETALA1, two central regulators of floral meristem identity. Expression patterns of the PUCHI and BOP genes point to a role in spatial control of flower-specific activation of these meristem identity genes.     

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.

sRNA, PARE, and AGO-IP sequencing uncovered the role of miRNAs during pollen development, showing that miRNAs transition from regulating genes involved in development to transposable elements.     

Identification of Most Stable Endogenous Control Genes for MicroRNA Quantification in the Developing Mouse Lung

MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies.