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The objective of this study was to evaluated fermentation losses and silage quality with the addition of different by-products in TMR. A completely randomized design was used with four treatments: Control (CTL, with hay, corn silage, cracked corn, soybean meal, wheat bran and minerals); CTL plus the addition of fresh orange pulp (FOP); CTL plus soybean hulls (SBH) or CTL plus wet brewery wastes (WBW), with six replicates. The ingredients were mixed and ensiled in 24 PVC experimental silos. After 56 days of ensiling, fermentation losses (effluents, dry matter and gases), and the TMR silage pH and chemical composition were analyzed; aerobic stability was evaluated over seven days after the silos were opened. Lower dry matter (P ? 0.0001) was observed in TMR ensiled with wet (WBW and FOP) compared to dry (SBH) by-products. Greater ADF (P = 0.031) was observed in SBH when compared to others by-products. WBW and FOP increased dry matter (P ? 0.0001) and effluent losses (P ? 0.0001) when compared to CTL and SBH. SBH treatment remained stable after 176-hour, which differ (P = 0.0015) compared with the other treatments (range from 94.5 to 99 hours of exposure to air). The density (kg DM/m3) changed (P ? 0.0001) among treatments. The inclusion of SBH in TMR silages had the greatest density, followed by CTL, WBW and FOP. The density was negatively correlated with DM losses ( r= - 0,81; P ? 0.0001). The pH differ (P = 0.003) among treatments. Highest pH of TMR silage was observed for SBH (3,67) and the lowest (3,56) for FOP. Soybean hulls successful improved aerobic stability and density, whereas aerobic stability and density were decreased and fermentation losses increased when fresh orange pulp and wet brewery were used. Little effects on chemical composition were observed, thereby agro-industrial by-products can be used in TMR silages as long as there is adequate product availability and pricing.

Object: Surgical training is often seen as very timely and cost consuming as it requires multiple interactions with other experienced physicians. Through the use of video motion capture (VMC), novice surgeon's skills will be compared objectively to those of more experienced surgeons. Methods: VMC was used to capture the movements of four neurosurgery residents performing five simple tasks: (1) threading the needle through the provided plastic vessel; (2) pulling the needle through the provided plastic vessel; and (3,4,5) tying the suture three times. Results: It was concluded that experienced subjects recorded more accurate and precise motions within a shorter amount of time when compared to novice subjects. There was a decrease in time, elapsed path, and thumb tip distance with increasing experience. Conclusions: The use of VMC proves to be a successful way to compare the differences between different levels of surgical expertise and we hope that this research will impact training paradigms for future surgical trainees. Keywords: Neurology, Neurosurgery, Medical education, Surgical training, Human motion, Motion capture

INTRODUCCIÓN: La reabsorción radicular asociada al tratamiento ortodóncico es un fenómeno, que aunque ha sido bastante debatido en la literatura, su naturaleza, causa y efectos son aún controvertidos. Debido a la discusión que se presenta en la literatura acerca del comportamiento radicular durante el tiempo del tratamiento de ortodoncia, se realizó un estudio de seguimiento radiográfico con el propósito de determinar cuantitativamente los cambios en la anatomía radicular considerando intervalos fijos de tiempo (0, 6, 12, 18 meses). MÉTODOS: el análisis se centró en la evaluación de variables relacionadas con la reabsorción radicular, incluyendo variables dependientes como: área y longitud radicular e independientes como: tiempo de tratamiento, género, edad, forma radicular, tipo de alambre utilizado, diagnóstico, hábitos, trauma, exodoncias, antecedentes médicos, expansión maxilar, uso de elásticos y tipo de brackets. La técnica usada para evaluar los cambios radiculares radiográficos fue la morfometría digital. Se tomó una muestra de 26 pacientes (14 hombres y 12 mujeres) entre los 11 y 21 años, atendidos por estudiantes del posgrado de Odontología Integral del Adolescente y Ortodoncia de la Facultad de Odontología de la Universidad de Antioquia. RESULTADOS Y CONCLUSIONES: los resultados obtenidos con significancia estadística a un nivel de confianza del 95% permitieron las siguientes conclusiones: 1) Se observaron cambios en la integridad radicular en todos los incisivos superiores durante los 18 meses de tratamiento. 2) Los mayores cambios se presentaron durante los primeros seis meses de tratamiento y fueron significativos a los 18 meses. 3) Los incisivos laterales superiores fueron más susceptibles a la reabsorción radicular que los incisivos centrales superiores. 4) Las condiciones que favorecieron la reabsorción radicular en esta muestra fueron: género masculino, antecedente médico de rinitis, expansión maxilar previa a la terapia ortodóncica, tratamiento ortodóncico con exodoncias de primeros bicúspides superiores y aparatología con prescripción estándar. 5) Los resultados discutidos son aplicables a la muestra estudiada y no se deberán extraplolar a todo el universo, pero la información si es muy valiosa para considerarse en futuros estudios con muestra mayor.INTRODUCTION: orthodontically induced external root resorption is not a well understood phenomenon, and although it has been widely studied in the orthodontic literature, its nature, causes and effects are still controversial. Since there is no clarity about the apical root changes that take place during the orthodontic treatment, the aim of this study was to determine quantitative changes in root morphology during fixed periods of time (0, 6, 12, 18 months) based on a radiographic assessment. METHODS: the dependent variables analysed were: area and root length, the independent variables were. treatment time, gender, age, root form, type of wire, diagnosis, oral habits, trauma, extractions, medical history, maxillary expansion and use of elastics. The method used to evaluate and calculate the root changes was the digital morphometric analysis of periapical radiographs. The sample consisted of 26 young patients (14 males- 12 females) between 11-21 years of age that were going to be treated at the Dental School of the University of Antioquia by students of the postgraduate Orthodontic program. RESULTS AND CONCLUSIONS: the results had a statistical significance of 95% and led to the following conclusions: 1) root integrity was affected in the upper incisors of all patients during the 18 months of treatment. 2) The greatest changes occurred during the first six months and were significant after 18 months of treatment. 3) Upper lateral incisors were more susceptible to root resorption than upper central incisors. 4) The conditions that favored root resorption in this sample were: male gender, medical history of respiratory allergies, maxillary expansion prior to orthodontic therapy, orthodontic treatment with extractions of first upper bicuspids, and standard edgewise brackets. 5) These results are applicable to the sample analyzed and should not be extrapolated to the general population, but the information is valid to be considered in future studies with a larger sample.

Formation of functional pollen and successful fertilization rely on the spatial and temporal regulation of anther and pollen development. This process responds to environmental cues to maintain optimal fertility despite climatic changes. Arabidopsis transcription factors basic helix-loop-helix (bHLH) 10, 89, and 91 were previously thought to be functionally redundant in their control of male reproductive development, however here we show that they play distinct roles in the integration of light signals to maintain pollen development under different environmental conditions. Combinations of the double and triple bHLH10,89,91 mutants were analysed under normal (200 μmol m-2 s-1) and low (50 μmol m-2 s-1) light conditions to determine the impact on fertility. Transcriptomic analysis of a new conditionally sterile bhlh89,91 double mutant shows differential regulation of genes related to sexual reproduction, hormone signal transduction, and lipid storage and metabolism under low light. Here we have shown that bHLH89 and bHLH91 play a role in regulating fertility in response to light, suggesting that they function in mitigating environmental variation to ensure fertility is maintained under environmental stress., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The anther tapetum helps control microspore release and essential components for pollen wall formation. TAPETAL DEVELOPMENT and FUNCTION1 (TDF1) is an essential R2R3 MYB tapetum transcription factor in Arabidopsis thaliana; however, little is known about pollen development in the temperate monocot barley. Here, we characterize the barley (Hordeum vulgare L.) TDF1 ortholog using reverse genetics and transcriptomics. Spatial/temporal expression analysis indicates HvTDF1 has tapetum-specific expression during anther stage 7/8. Homozygous barley hvtdf1 mutants exhibit male sterility with retarded tapetum development, delayed tapetum endomitosis and cell wall degeneration, resulting in enlarged, vacuolated tapetum surrounding collapsing microspores. Transient protein expression and dual-luciferase assays show TDF1 is a nuclear-localized, transcription activator, that directly activates osmotin proteins. Comparison of hvtdf1 transcriptome data revealed several pathways were delayed, endorsing the observed retarded anther morphology. Arabidopsis tdf1 mutant fertility was recovered by HvTDF1, supporting a conserved role for TDF1 in monocots and dicots. This indicates that tapetum development shares similarity between monocot and dicots; however, barley HvTDF1 appears to uniquely act as a modifier to activate tapetum gene expression pathways, which are subsequently also induced by other factors. Therefore, the absence of HvTDF1 results in delayed developmental progression rather than pathway failure, although inevitably still results in pollen degeneration., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Rising temperatures and extreme heat events threaten rice production. Half of the global population relies on rice for basic nutrition, and therefore developing heat-tolerant rice is essential. During vegetative development, reduced photosynthetic rates can limit growth and the capacity to store soluble carbohydrates. The photosystem II (PSII) complex is a particularly heat-labile component of photosynthesis. We have developed a high-throughput chlorophyll fluorescence-based screen for photosynthetic heat tolerance capable of screening hundreds of plants daily. Through measuring the response of maximum PSII efficiency to increasing temperature, this platform generates data for modelling the PSII-temperature relationship in large populations in a small amount of time. Coefficients from these models (photosynthetic heat tolerance traits) demonstrated high heritabilities across African (Oryza glaberrima) and Asian (Oryza sativa, Bengal Assam Aus Panel) rice diversity sets, highlighting valuable genetic variation accessible for breeding. Genome-wide association studies were performed across both species for these traits, representing the first documented attempt to characterize the genetic basis of photosynthetic heat tolerance in any species to date. A total of 133 candidate genes were highlighted. These were significantly enriched with genes whose predicted roles suggested influence on PSII activity and the response to stress. We discuss the most promising candidates for improving photosynthetic heat tolerance in rice., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Heat stress has a deleterious effect on male fertility in rice (Oryza sativa), but mechanisms to protect against heat stress in rice male gametophytes are poorly understood. Here, we have isolated and characterized a heat-sensitive male-sterile rice mutant, heat shock protein60-3b (oshsp60-3b), that shows normal fertility at optimal temperatures but decreasing fertility as temperatures increase. High temperatures interfered with pollen starch granule formation and reactive oxygen species (ROS) scavenging in oshsp60-3b anthers, leading to cell death and pollen abortion. In line with the mutant phenotypes, OsHSP60-3B was rapidly upregulated in response to heat shock and its protein products were localized to the plastid. Critically, overexpression of OsHSP60-3B enhanced the heat tolerance of pollen in transgenic plants. We demonstrated that OsHSP60-3B interacted with FLOURY ENDOSPERM6(FLO6) in plastids, a key component involved in the starch granule formation in the rice pollen. Western blot results showed that FLO6 level was substantially decreased in oshsp60-3b anthers at high temperature, indicating that OsHSP60-3B is required to stabilize FLO6 when temperatures exceed optimal conditions. We suggest that in response to high temperature, OsHSP60-3B interacts with FLO6 to regulate starch granule biogenesis in rice pollen and attenuates ROS levels in anthers to ensure normal male gametophyte development in rice., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Poaceae, or grasses, include many agriculturally important cereal crops such as rice ( Oryza sativa ), maize ( Zea mays ), barley ( Hordeum vulgare ) and bread wheat ( Triticum aestivum ). Barley is a widely grown cereal crop used for stock feed, malting and brewing. Abiotic stresses, particularly global warming, are the major causes of crop yield losses by affecting fertility and seed set. However, effects of heat stress on reproductive structures and fertility in barley have not been extensively investigated. In this study we examined three commercial European spring barley varieties under high temperature conditions to investigate the effects on floret development. Using a combination of fertility assays, X-ray micro computed tomography, 3-dimensional modelling, cytology and immunolabelling, we observed that male reproductive organs are severely impacted by increased temperature, while the female reproductive organs are less susceptible. Importantly, the timing of stress relative to reproductive development had a significant impact on fertility in a cultivar-dependent manner, this was most significant at pollen mitosis stage with fertility ranged from 31.6-56.0% depending on cultivar. This work provides insight into how heat stress, when applied during male pollen mother cell meiosis and pollen mitosis, affects barley fertility and seed set, and also describes complementary invasive and non-invasive techniques to investigate floret development. This information will be used to identify and study barley cultivars that are less susceptible to heat stress at specific stages of floral development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Callens, Fernandez-Goméz, Tucker, Zhang and Wilson.)

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Key Message: Anther development and dehiscence is considered from an evolutionary perspective to identify drivers for differentiation, functional conservation and to identify key questions for future male reproduction research. Development of viable pollen and its timely release from the anther are essential for fertilisation of angiosperm flowers. The formation and subsequent dehiscence of the anther are under tight regulatory control, and these processes are remarkably conserved throughout the diverse families of the angiosperm clade. Anther development is a complex process, which requires timely formation and communication between the multiple somatic anther cell layers (the epidermis, endothecium, middle layer and tapetum) and the developing pollen. These layers go through regulated development and selective degeneration to facilitate the formation and ultimate release of the pollen grains. Insight into the evolution and divergence of anther development and dehiscence, especially between monocots and dicots, is driving greater understanding of the male reproductive process and increased, resilient crop yields. This review focuses on anther structure from an evolutionary perspective by highlighting their diversity across plant species. We summarise new findings that illustrate the complexities of anther development and evaluate how they challenge established models of anther form and function, and how they may help to deliver future sustainable crop yields., (© 2021. The Author(s).)

Impaired carbon metabolism and reproductive development constrain crop productivity during heat stress. Reproductive development is energy intensive, and its requirement for respiratory substrates rises as associated metabolism increases with temperature. Understanding how these processes are integrated and the extent to which they contribute to the maintenance of yield during and following periods of elevated temperatures is important for developing climate-resilient crops. Recent studies are beginning to demonstrate links between processes underlying carbon dynamics and reproduction during heat stress, consequently a summation of research that has been reported thus far and an evaluation of purported associations are needed to guide and stimulate future research. To this end, we review recent studies relating to source-sink dynamics, non-foliar photosynthesis and net carbon gain as pivotal in understanding how to improve reproductive development and crop productivity during heat stress. Rapid and precise phenotyping during narrow phenological windows will be important for understanding mechanisms underlying these processes, thus we discuss the development of relevant high-throughput phenotyping approaches that will allow for more informed decision-making regarding future crop improvement., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

A key target for the improvement of Oryza sativa (rice) is the development of heat-tolerant varieties. This necessitates the development of high-throughput methodologies for the screening of heat tolerance. Progress has been made to this end via visual scoring and chlorophyll fluorescence; however, these approaches demand large infrastructural investments to expose large populations of adult plants to heat stress. To address this bottleneck, we investigated the response of the maximum quantum efficiency of photosystem II (PSII) to rapidly increasing temperatures in excised leaf segments of juvenile rice plants. Segmented models explained the majority of the observed variation in response. Coefficients from these models, i.e. critical temperature (T crit ) and the initial response (m 1 ), were evaluated for their usability for forecasting adult heat tolerance, measured as the vegetative heat tolerance of adult rice plants through visual (stay-green) and chlorophyll fluorescence (ɸPSII) approaches. We detected substantial variation in heat tolerance of a randomly selected set of indica rice varieties. Both T crit and m 1 were associated with measured heat tolerance in adult plants, highlighting their usability as high-throughput proxies. Variation in heat tolerance was associated with daytime respiration but not with photosynthetic capacity, highlighting a role for the non-photorespiratory release of CO 2 in heat tolerance. To date, this represents the first published instance of genetic variation in these key gas-exchange traits being quantified in response to heat stress in a diverse set of rice accessions. These results outline an efficient strategy for screening heat tolerance and accentuate the need to focus on reduced rates of respiration to improve heat tolerance in rice., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Understanding the control of fertility is critical for crop yield and breeding; this is particularly important for hybrid breeding to capitalize upon the resultant hybrid vigour. Different hybrid breeding systems have been adopted; however, these are challenging and crop specific. Mutants with environmentally reversible fertility offer valuable opportunities for hybrid breeding. The barley HvMS1 gene encodes a PHD-finger transcription factor that is expressed in the anther tapetum, which is essential for pollen development and causes complete male sterility when overexpressed in barley. This male sterility is due at least in part to indehiscent anthers resulting from incomplete tapetum degeneration, failure of anther opening, and sticky pollen under normal growth conditions (15 °C). However, dehiscence and fertility are restored when plants are grown at temperatures >20 °C, or when transferred to >20 °C during flowering prior to pollen mitosis I, with transfer at later stages unable to rescue fertility in vivo. As far as we are aware, this is the first report of thermosensitive male sterility in barley. This offers opportunities to understand the impact of temperature on pollen development and potential applications for environmentally switchable hybrid breeding systems; it also provides a 'female' male-sterile breeding tool that does not need emasculation to facilitate backcrossing., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Sporophytic pollen coat proteins (sPCPs) derived from the anther tapetum are deposited into pollen wall cavities and function in pollen-stigma interactions, pollen hydration, and environmental protection. In Arabidopsis, 13 highly abundant proteins have been identified in pollen coat, including seven major glycine-rich proteins GRP14, 16, 17, 18, 19, 20, and GRP-oleosin; two caleosin-related family proteins (AT1G23240 and AT1G23250); three lipase proteins EXL4, EXL5 and EXL6, and ATA27/BGLU20. Here, we show that GRP14, 17, 18, 19, and EXL4 and EXL6 fused with green fluorescent protein (GFP) are translated in the tapetum and then accumulate in the anther locule following tapetum degeneration. The expression of these sPCPs is dependent on two essential tapetum transcription factors, MALE STERILE188 (MS188) and MALE STERILITY 1 (MS1). The majority of sPCP genes are up-regulated within 30 h after MS1 induction and could be restored by MS1 expression driven by the MS188 promoter in ms188, indicating that MS1 is sufficient to activate their expression; however, additional MS1 downstream factors appear to be required for high-level sPCP expression. Our ChIP, in vivo transactivation assay, and EMSA data indicate that MS188 directly activates MS1. Together, these results reveal a regulatory cascade whereby outer pollen wall formation is regulated by MS188 followed by synthesis of sPCPs controlled by MS1., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Wheat is one of the most important crops in the world; however, loss of genetic variability and abiotic stress caused by variable climatic conditions threaten future productivity. Reproduction is critical for wheat yield; however, pollen development is amongst the developmental stages most sensitive to stresses such as heat, cold, or drought. A better understanding of how anther and pollen development is regulated is needed to help produce more resilient crops and ensure future yield increases. However, in cereals such as wheat, barley, and rice, flowers form within the developing pseudostem and therefore accurate staging of floral materials is extremely challenging. This makes detailed phenotypic and molecular analysis of floral development very difficult, particularly when limited plant material is available, for example with mutant or transgenic lines. Here we present an accurate approach to overcome this problem, by non-destructive staging of reproduction development in Cadenza, the widely used spring wheat research variety. This uses a double-scale system whereby anther and pollen development can be predicted in relation to spike size and spike position within the pseudostem. This system provides an easy, reproducible method that facilitates accurate sampling and analysis of floral materials, to enable anther and pollen developmental research., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

A well-defined set of regulatory pathways control entry into the reproductive phase in flowering plants, but little is known about the mechanistic control of the end-of-flowering despite this being a critical process for optimization of fruit and seed production. Complete fruit removal, or lack of fertile fruit-set, prevents timely inflorescence arrest in Arabidopsis, leading to a previous proposal that a cumulative fruit/seed-derived signal causes simultaneous 'global proliferative arrest'. Recent studies have suggested that inflorescence arrest involves gene expression changes in the inflorescence meristem that are, at least in part, controlled by the FRUITFULL-APETALA2 pathway; however, there is limited understanding of how this process is coordinated at the whole-plant level. Here, we provide a framework for the communication previously inferred in the global proliferative arrest model. We show that the end-of-flowering in Arabidopsis is not 'global' and does not occur synchronously between branches, but rather that the arrest of each inflorescence is a local process, driven by auxin export from fruit proximal to the inflorescence apex. Furthermore, we show that inflorescences are competent for arrest only once they reach a certain developmental age. Understanding the regulation of inflorescence arrest will be of major importance to extending and maximizing crop yields.

ROOT UV-B SENSITIVE4 (RUS4) encodes a protein with no known function that contains a conserved Domain of Unknown Function 647 (DUF647). The DUF647-containing proteins RUS1 and RUS2 have previously been associated with root UV-B-sensing pathway that plays a major role in Arabidopsis early seedling morphogenesis and development. Here, we show that RUS4 knockdown Arabidopsis plants, referred to as amiR-RUS4, were severely reduced in male fertility with indehiscent anthers. Light microscopy of anther sections revealed a significantly reduced secondary wall thickening in the endothecium of amiR-RUS4 anthers. We further show that the transcript abundance of the NAC domain genes NAC SECONDARY WALL THICKENING PROMOTING FACTOR1 (NST1) and NST2, which have been shown to regulate the secondary cell wall thickenings in the anther endothecium, were dramatically reduced in the amiR-RUS4 floral buds. Expression of the secondary cell wall-associated MYB transcription factor genes MYB103 and MYB85 were also strongly reduced in floral buds of the amiR-RUS4 plants. Overexpression of RUS4 led to increased secondary thickening in the endothecium. However, the rus4-2 mutant exhibited no obvious phenotype. Promoter-GUS analysis revealed that the RUS4 promoter was highly active in the anthers, supporting its role in anther development. Taken together, these results suggest that RUS4, probably functions redundantly with other genes, may play an important role in the secondary thickening formation in the anther endothecium by indirectly affecting the expression of secondary cell wall biosynthetic genes., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Key Message: Linear modelling approaches detected significant gradients in organ growth and patterning across early flowers of the Arabidopsis inflorescence and uncovered evidence of new roles for gibberellin in floral development. Most flowering plants, including the genetic model Arabidopsis thaliana, produce multiple flowers in sequence from a reproductive shoot apex to form a flower spike (inflorescence). The development of individual flowers on an Arabidopsis inflorescence has typically been considered as highly stereotypical and uniform, but this assumption is contradicted by the existence of mutants with phenotypes visible in early flowers only. This phenomenon is demonstrated by mutants partially impaired in the biosynthesis of the phytohormone gibberellin (GA), in which floral organ growth is retarded in the first flowers to be produced but has recovered spontaneously by the 10th flower. We presently lack systematic data from multiple flowers across the Arabidopsis inflorescence to explain such changes. Using mutants of the GA 20-OXIDASE (GA20ox) GA biosynthesis gene family to manipulate endogenous GA levels, we investigated the dynamics of changing floral organ growth across the early Arabidopsis inflorescence (flowers 1-10). Modelling of floral organ lengths identified a significant, GA-independent gradient of increasing stamen length relative to the pistil in the wild-type inflorescence that was separable from other, GA-dependent effects. It was also found that the first flowers exhibited unstable organ patterning in contrast to later flowers and that this instability was prolonged by exogenous GA treatment. These findings indicate that the development of individual flowers is influenced by hitherto unknown factors acting across the inflorescence and also suggest novel functions for GA in floral patterning.

Many monocot plants have high social and economic value. These include grasses such as rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare), which produce soft commodities for many food and beverage industries, and ornamental flowers such ase lily (Lilium longiflorum) and orchid (Oncidium Gower Ramsey), which represent an important component of international flower markets. There is constant pressure to improve the development and diversity of these species, with a significant emphasis on flower development, and this is particularly relevant considering the impact of changing environments on reproduction and thus yield. MADS-box proteins are a family of transcription factors that contain a conserved 60 amino acid MADS-box motif. In plants, attention has been devoted to characterization of this family due to their roles in inflorescence and flower development, which holds promise for the modification of floral architecture for plant breeding. This has been explored in diverse angiosperms, but particularly the dicot model Arabidopsis thaliana. The focus of this review is on the less well characterized roles of the MADS-box proteins in monocot flower development and how changes in MADS-box proteins throughout evolution may have contributed to creating a diverse range of flowers. Examining these changes within the monocots can identify the importance of certain genes and pinpoint those which might be useful in future crop improvement and breeding strategies.

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., (© 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.)

Mitochondrial uncoupling proteins (UCPs) sustain mitochondrial respiration independent of intracellular ATP concentration. Uncoupled respiration is particularly beneficial under stress conditions, during which both photosynthesis and respiration may be impaired. Sustaining carbon fixation during the reproductive phase is essential for plants to develop viable pollen grains and for seed setting. Here, we examined whether UCP1 overexpression (UCP1-oe) would help tobacco plants cope with drought stress during reproductive development. We observed that WT and UCP1-oe plants lost water at the same rate under moderate drought stress, but that UCP1-oe lines regained water faster upon rewatering. UCP1-oe plants maintained higher levels of respiration and photosynthesis and decreased H 2 O 2 content in the leaves during the drought stress period. We examined whether UCP1-oe impacts reproductive tissues and seed production by monitoring the progress of flower development, focusing on the early stages of pollen formation. UCP1-oe lines induced the expression of mitochondrial genes and increased mtDNA content in reproductive tissues, which increased the consumption of carbohydrates and reduced H 2 O 2 content and pollen disturbances. Finally, the beneficial impact of UCP1-oe on the source and sink organs resulted in an increased seed size and number under both control conditions and drought stress.

Successful fertilization relies on the production and effective release of viable pollen. Failure of anther opening (dehiscence), results in male sterility, although the pollen may be fully functional. MYB26 regulates the formation of secondary thickening in the anther endothecium, which is critical for anther dehiscence and fertility. Here, we show that although the MYB26 transcript shows expression in multiple floral organs, the MYB26 protein is localized specifically to the anther endothecium nuclei and that it directly regulates two NAC domain genes, NST1 and NST2 , which are critical for the induction of secondary thickening biosynthesis genes. However, there is a complex relationship of regulation between these genes and MYB26. Using DEX-inducible MYB26 lines and overexpression in the various mutant backgrounds, we have shown that MYB26 up-regulates both NST1 and NST2 expression. Surprisingly normal thickening and fertility rescue does not occur in the absence of MYB26, even with constitutively induced NST1 and NST2 , suggesting an additional essential role for MYB26 in this regulation. Combined overexpression of NST1 and NST2 in myb26 facilitates limited ectopic thickening in the anther epidermis, but not in the endothecium, and thus fails to rescue dehiscence. Therefore, by a series of regulatory controls through MYB26, NST1, NST2, secondary thickening is formed specifically within the endothecium; this specificity is essential for anther opening., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Background: Accurate floral staging is required to aid research into pollen and flower development, in particular male development. Pollen development is highly sensitive to stress and is critical for crop yields. Research into male development under environmental change is important to help target increased yields. This is hindered in monocots as the flower develops internally in the pseudostem. Floral staging studies therefore typically rely on destructive analysis, such as removal from the plant, fixation, staining and sectioning. This time-consuming analysis therefore prevents follow up studies and analysis past the point of the floral staging., Results: This study focuses on using X-ray µCT scanning to allow quick and detailed non-destructive internal 3D phenotypic information to allow accurate staging of Arabidopsis thaliana L. and Barley ( Hordeum vulgare L.) flowers. X-ray µCT has previously relied on fixation methods for above ground tissue, therefore two contrast agents (Lugol's iodine and Bismuth) were observed in Arabidopsis and Barley in planta to circumvent this step. 3D models and 2D slices were generated from the X-ray µCT images providing insightful information normally only available through destructive time-consuming processes such as sectioning and microscopy. Barley growth and development was also monitored over three weeks by X-ray µCT to observe flower development in situ. By measuring spike size in the developing tillers accurate non-destructive staging at the flower and anther stages could be performed; this staging was confirmed using traditional destructive microscopic analysis., Conclusion: The use of X-ray micro computed tomography (µCT) scanning of living plant tissue offers immense benefits for plant phenotyping, for successive developmental measurements and for accurate developmental timing for scientific measurements. Nevertheless, X-ray µCT remains underused in plant sciences, especially in above-ground organs, despite its unique potential in delivering detailed non-destructive internal 3D phenotypic information. This work represents a novel application of X-ray µCT that could enhance research undertaken in monocot species to enable effective non-destructive staging and developmental analysis for molecular genetic studies and to determine effects of stresses at particular growth stages.

Viable pollen is essential for plant reproduction and crop yield. Its production requires coordinated expression at specific stages during anther development, involving early meiosis-associated events and late pollen wall formation. The ABORTED MICROSPORES (AMS) transcription factor is a master regulator of sporopollenin biosynthesis, secretion and pollen wall formation in Arabidopsis. Here we show that it has complex regulation and additional essential roles earlier in pollen formation. An inducible-AMS reporter was created for functional rescue, protein expression pattern analysis, and to distinguish between direct and indirect targets. Mathematical modelling was used to create regulatory networks based on wild-type RNA and protein expression. Dual activity of AMS was defined by biphasic protein expression in anther tapetal cells, with an initial peak around pollen meiosis and then later during pollen wall development. Direct AMS-regulated targets exhibit temporal regulation, indicating that additional factors are associated with their regulation. We demonstrate that AMS biphasic expression is essential for pollen development, and defines distinct functional activities during early and late pollen development. Mathematical modelling suggests that AMS may competitively form a protein complex with other tapetum-expressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Pollen development is a critical step in plant development that is needed for successful breeding and seed formation. Manipulation of male fertility has proved a useful trait for hybrid breeding and increased crop yield. However, although there is a good understanding developing of the molecular mechanisms of anther and pollen anther development in model species, such as Arabidopsis and rice, little is known about the equivalent processes in important crops. Nevertheless the onset of increased genomic information and genetic tools is facilitating translation of information from the models to crops, such as barley and wheat; this will enable increased understanding and manipulation of these pathways for agricultural improvement., (© 2015 Institute of Botany, Chinese Academy of Sciences.)

Key Message: Transgenic Lilium lines have been generated by Agrobacterium -mediated transformation that have enhanced resistance to Botrytis cinerea as a consequence of ectopic expression of a rice chitinase gene. The production of ornamentals is an important global industry, with Lilium being one of the six major bulb crops in the world. The international trade in ornamentals is in the order of £60-75 billion and is expected to increase worldwide by 2-4% per annum. The continued success of the floriculture industry depends on the introduction of new species/cultivars with major alterations in key agronomic characteristics, such as resistance to pathogens. Fungal diseases are the cause of reduced yields and marketable quality of cultivated plants, including ornamental species. The fungal pathogen Botrytis causes extreme economic losses to a wide range of crop species, including ornamentals such as Lilium. Agrobacterium-mediated transformation was used to develop Lilium oriental cv. 'Star Gazer' plants that ectopically overexpress the Rice Chitinase 10 gene (RCH10), under control of the CaMV35S promoter. Levels of conferred resistance linked to chitinase expression were evaluated by infection with Botrytis cinerea; sporulation was reduced in an in vitro assay and the relative expression of the RCH10 gene was determined by quantitative reverse transcriptase-PCR. The extent of resistance to Botrytis, compared to that of the wild type plants, showed a direct correlation with the level of chitinase gene expression. Transgenic plants grown to flowering showed no detrimental phenotypic effects associated with transgene expression. This is the first report of Lilium plants with resistance to Botrytis cinerea generated by a transgenic approach.

Floral formation, in particular anther and pollen development, is a complex biological process with critical importance for seed set and for targeted plant breeding. Many key transcription factors regulating this process have been identified; however, their direct role remains largely unknown. Using publicly available gene expression data from Arabidopsis (Arabidopsis thaliana), focusing on those studies that analyze stamen-, pollen-, or flower-specific expression, we generated a network model of the global transcriptional interactions (FlowerNet). FlowerNet highlights clusters of genes that are transcriptionally coregulated and therefore likely to have interacting roles. Focusing on four clusters, and using a number of data sets not included in the generation of FlowerNet, we show that there is a close correlation in how the genes are expressed across a variety of conditions, including male-sterile mutants. This highlights the important role that FlowerNet can play in identifying new players in anther and pollen development. However, due to the use of general floral expression data in FlowerNet, it also has broad application in the characterization of genes associated with all aspects of floral development and reproduction. To aid the dissection of genes of interest, we have made FlowerNet available as a community resource (http://www.cpib.ac.uk/anther). For this resource, we also have generated plots showing anther/flower expression from a variety of experiments: These are normalized together where possible to allow further dissection of the resource., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Soybean [Glycine max (L.) Merr.] is one of the world's major crops, and soybean seeds are a rich and important resource for proteins and oils. While "omics" studies, such as genomics, transcriptomics, and proteomics, have been widely applied in soybean molecular research, fewer metabolomic studies have been conducted for large-scale detection of low molecular weight metabolites, especially in soybean seeds. In this study, we investigated the seed metabolomes of 29 common soybean cultivars through combined gas chromatography-mass spectrometry and ultra-performance liquid chromatography-tandem mass spectrometry. One hundred sixty-nine named metabolites were identified and subsequently used to construct a metabolic network of mature soybean seed. Among the 169 detected metabolites, 104 were found to be significantly variable in their levels across tested cultivars. Metabolite markers that could be used to distinguish genetically related soybean cultivars were also identified, and metabolite-metabolite correlation analysis revealed some significant associations within the same or among different metabolite groups. Findings from this work may potentially provide the basis for further studies on both soybean seed metabolism and metabolic engineering to improve soybean seed quality and yield., (© 2014 Institute of Botany, Chinese Academy of Sciences.)

Controlling pollen development is of major commercial importance in generating hybrid crops and selective breeding, but characterized genes for male sterility in crops are rare, with no current examples in barley. However, translation of knowledge from model species is now providing opportunities to understand and manipulate such processes in economically important crops. We have used information from regulatory networks in Arabidopsis to identify and functionally characterize a barley PHD transcription factor MALE STERTILITY1 (MS1), which expresses in the anther tapetum and plays a critical role during pollen development. Comparative analysis of Arabidopsis, rice and Brachypodium genomes was used to identify conserved regions in MS1 for primer design to amplify the barley MS1 gene; RACE-PCR was subsequently used to generate the full-length sequence. This gene shows anther-specific tapetal expression, between late tetrad stage and early microspore release. HvMS1 silencing and overexpression in barley resulted in male sterility. Additionally, HvMS1 cDNA, controlled by the native Arabidopsis MS1 promoter, successfully complemented the homozygous ms1 Arabidopsis mutant. These results confirm the conservation of MS1 function in higher plants and in particular in temperate cereals. This has provided the first example of a characterized male sterility gene in barley, which presents a valuable tool for the future control of male fertility in barley for hybrid development., (© 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning., (© 2014 American Society of Plant Biologists. All rights reserved.)

The trans-Golgi network (TGN) plays a central role in cellular secretion and has been implicated in sorting cargo destined for the plasma membrane. Previously, the Arabidopsis (Arabidopsis thaliana) echidna (ech) mutant was shown to exhibit a dwarf phenotype due to impaired cell expansion. However, ech also has a previously uncharacterized phenotype of reduced male fertility. This semisterility is due to decreased anther size and reduced amounts of pollen but also to decreased pollen viability, impaired anther opening, and pollen tube growth. An ECH translational fusion (ECHPro:ECH-yellow fluorescent protein) revealed developmentally regulated tissue-specific expression, with expression in the tapetum during early anther development and microspore release and subsequent expression in the pollen, pollen tube, and stylar tissues. Pollen viability and production, along with germination and pollen tube growth, were all impaired. The ech anther endothecium secondary wall thickening also appeared reduced and disorganized, resulting in incomplete anther opening. This did not appear to be due to anther secondary thickening regulatory genes but perhaps to altered secretion of wall materials through the TGN as a consequence of the absence of the ECH protein. ECH expression is critical for a variety of aspects of male reproduction, including the production of functional pollen grains, their effective release, germination, and tube formation. These stages of pollen development are fundamentally influenced by TGN trafficking of hormones and wall components. Overall, this suggests that the fertility defect is multifaceted, with the TGN trafficking playing a significant role in the process of both pollen formation and subsequent fertilization.

Excessive gibberellin (GA) signalling, mediated through the DELLA proteins, has a negative impact on plant fertility. Loss of DELLA activity in the monocot rice (Oryza sativa) causes complete male sterility, but not in the dicot model Arabidopsis (Arabidopsis thaliana) ecotype Landsberg erecta (Ler), in which DELLA function has been studied most extensively, leading to the assumption that DELLA activity is not essential for Arabidopsis pollen development. A novel DELLA fertility phenotype was identified in the Columbia (Col-0) ecotype that necessitates re-evaluation of the general conclusions drawn from Ler. Fertility phenotypes were compared between the Col-0 and Ler ecotypes under conditions of chemical and genetic GA overdose, including mutants in both ecotypes lacking the DELLA paralogues REPRESSOR OF ga1-3 (RGA) and GA INSENSITIVE (GAI). Ler displays a less severe fertility phenotype than Col-0 under GA treatment. Col-0 rga gai mutants, in contrast with the equivalent Ler phenotype, were entirely male sterile, caused by post-meiotic defects in pollen development, which were rescued by the reintroduction of DELLA into either the tapetum or developing pollen. We conclude that DELLA activity is essential for Arabidopsis pollen development. Differences between the fertility responses of Col-0 and Ler might be caused by differences in downstream signalling pathways or altered DELLA expression., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Programmed cell death is essential for the development of multicellular organisms, yet pathways of plant programmed cell death and its regulation remain elusive. Here we report that ETERNAL TAPETUM 1, a basic helix-loop-helix transcription factor conserved in land plants, positively regulates programmed cell death in tapetal cells in rice anthers. eat1 exhibits delayed tapetal cell death and aborted pollen formation. ETERNAL TAPETUM 1 directly regulates the expression of OsAP25 and OsAP37, which encode aspartic proteases that induce programmed cell death in both yeast and plants. Expression and genetic analyses revealed that ETERNAL TAPETUM 1 acts downstream of TAPETUM DEGENERATION RETARDATION, another positive regulator of tapetal programmed cell death, and that ETERNAL TAPETUM 1 can also interact with the TAPETUM DEGENERATION RETARDATION protein. This study demonstrates that ETERNAL TAPETUM 1 promotes aspartic proteases triggering plant programmed cell death, and reveals a dynamic regulatory cascade in male reproductive development in rice.

Understanding the processes that underlie pollen release is a prime target for controlling fertility to enable selective breeding and the efficient production of hybrid crops. Pollen release requires anther opening, which involves changes in the biomechanical properties of the anther wall. In this research, we develop and use a mathematical model to understand how these biomechanical processes lead to anther opening. Our mathematical model describing the biomechanics of anther opening incorporates the bilayer structure of the mature anther wall, which comprises the outer epidermal cell layer, whose turgor pressure is related to its hydration, and the endothecial layer, whose walls contain helical secondary thickening, which resists stretching and bending. The model describes how epidermal dehydration, in association with the thickened endothecial layer, creates forces within the anther wall causing it to bend outwards, resulting in anther opening and pollen release. The model demonstrates that epidermal dehydration can drive anther opening, and suggests why endothecial secondary thickening is essential for this process (explaining the phenotypes presented in the myb26 and nst1nst2 mutants). The research hypothesizes and demonstrates a biomechanical mechanism for anther opening, which appears to be conserved in many other biological situations where tissue movement occurs., (© 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.)

Background: Male reproduction is an essential biological event in the plant life cycle separating the diploid sporophyte and haploid gametophyte generations, which involves expression of approximately 20,000 genes. The control of male reproduction is also of economic importance for plant breeding and hybrid seed production. With the advent of forward and reverse genetics and genomic technologies, a large number of male reproduction-related genes have been identified. Thus it is extremely challenging for individual researchers to systematically collect, and continually update, all the available information on genes and mutants related to plant male reproduction. The aim of this study is to manually curate such gene and mutant information and provide a web-accessible resource to facilitate the effective study of plant male reproduction., Description: Plant Male Reproduction Database (PMRD) is a comprehensive resource for browsing and retrieving knowledge on genes and mutants related to plant male reproduction. It is based upon literature and biological databases and includes 506 male sterile genes and 484 mutants with defects of male reproduction from a variety of plant species. Based on Gene Ontology (GO) annotations and literature, information relating to a further 3697 male reproduction related genes were systematically collected and included, and using in text curation, gene expression and phenotypic information were captured from the literature. PMRD provides a web interface which allows users to easily access the curated annotations and genomic information, including full names, symbols, locations, sequences, expression patterns, functions of genes, mutant phenotypes, male sterile categories, and corresponding publications. PMRD also provides mini tools to search and browse expression patterns of genes in microarray datasets, run BLAST searches, convert gene ID and generate gene networks. In addition, a Mediawiki engine and a forum have been integrated within the database, allowing users to share their knowledge, make comments and discuss topics., Conclusion: PMRD provides an integrated link between genetic studies and the rapidly growing genomic information. As such this database provides a global view of plant male reproduction and thus aids advances in this important area.

A prerequisite to study the molecular genetic pathways of pollen and anther development is an accurate staging system for reproductive development. However in barley, floret formation occurs mainly within the pseudostem, which makes the observation of floret development and access to the floret particularly difficult without dissecting the plant. Thus selecting stages for molecular analysis cannot be done non-destructively. A staging method has therefore been developed for barley in order to define the relationship between readily detectable growth points and reproductive development, to provide a clear key to enable accurate selection of reproductive material. Initial staging followed the traditional Zadoks decimal system, with minor adaptations to stages 31-34 and stage 37. The later stages, from 37 onward, were replaced by growth staging based upon the last flag elongation (LFE) and the position occupied by the spike within the pseudostem. Spike size could be readily predicted by using the staging system incorporating Zadoks stages 31-37, supplemented with substages and by LFE staging to improve accuracy. The different spike sizes, as well as the LFE stages, showed a clear relationship to events occurring within the anther, as confirmed by light microscopy of the anthers. The defined relationship between spike size and development to anther development now makes possible the accurate prediction of anther and pollen progression by external staging. This, therefore, provides a mechanism for non-destructive selection of material for analysis that is critical for the molecular characterization of genes in anther and pollen development.

Protein interactions are fundamental to the molecular processes occurring within an organism and can be utilized in network biology to help organize, simplify, and understand biological complexity. Currently, there are more than 10 publicly available Arabidopsis (Arabidopsis thaliana) protein interaction databases. However, there are limitations with these databases, including different types of interaction evidence, a lack of defined standards for protein identifiers, differing levels of information, and, critically, a lack of integration between them. In this paper, we present an interactive bioinformatics Web tool, ANAP (Arabidopsis Network Analysis Pipeline), which serves to effectively integrate the different data sets and maximize access to available data. ANAP has been developed for Arabidopsis protein interaction integration and network-based study to facilitate functional protein network analysis. ANAP integrates 11 Arabidopsis protein interaction databases, comprising 201,699 unique protein interaction pairs, 15,208 identifiers (including 11,931 The Arabidopsis Information Resource Arabidopsis Genome Initiative codes), 89 interaction detection methods, 73 species that interact with Arabidopsis, and 6,161 references. ANAP can be used as a knowledge base for constructing protein interaction networks based on user input and supports both direct and indirect interaction analysis. It has an intuitive graphical interface allowing easy network visualization and provides extensive detailed evidence for each interaction. In addition, ANAP displays the gene and protein annotation in the generated interactive network with links to The Arabidopsis Information Resource, the AtGenExpress Visualization Tool, the Arabidopsis 1,001 Genomes GBrowse, the Protein Knowledgebase, the Kyoto Encyclopedia of Genes and Genomes, and the Ensembl Genome Browser to significantly aid functional network analysis. The tool is available open access at http://gmdd.shgmo.org/Computational-Biology/ANAP.

Gibberellin (GA) biosynthesis is necessary for normal plant development, with later GA biosynthetic stages being governed by multigene families. Arabidopsis thaliana contains five GA 20-oxidase (GA20ox) genes, and past work has demonstrated the importance of GA20ox1 and -2 for growth and fertility. Here, we show through systematic mutant analysis that GA20ox1, -2, and -3 are the dominant paralogs; their absence results in severe dwarfism and almost complete loss of fertility. In vitro analysis revealed that GA20ox4 has full GA20ox activity, but GA20ox5 catalyzes only the first two reactions of the sequence by which GA(12) is converted to GA(9). GA20ox3 functions almost entirely redundantly with GA20ox1 and -2 at most developmental stages, including the floral transition, while GA20ox4 and -5 have very minor roles. These results are supported by analysis of the gene expression patterns in promoter:β-glucuronidase reporter lines. We demonstrate that fertility is highly sensitive to GA concentration, that GA20ox1, -2, and -3 have significant effects on floral organ growth and anther development, and that both GA deficiency and overdose impact on fertility. Loss of GA20ox activity causes anther developmental arrest, with the tapetum failing to degrade. Some phenotypic recovery of late flowers in GA-deficient mutants, including ga1-3, indicated the involvement of non-GA pathways in floral development.

Total internal reflection fluorescence microscopy (TIRFM) has been proven to be an extremely powerful technique in animal cell research for generating high contrast images and dynamic protein conformation information. However, there has long been a perception that TIRFM is not feasible in plant cells because the cell wall would restrict the penetration of the evanescent field and lead to scattering of illumination. By comparative analysis of epifluorescence and TIRF in root cells, it is demonstrated that TIRFM can generate high contrast images, superior to other approaches, from intact plant cells. It is also shown that TIRF imaging is possible not only at the plasma membrane level, but also in organelles, for example the nucleus, due to the presence of the central vacuole. Importantly, it is demonstrated for the first time that this is TIRF excitation, and not TIRF-like excitation described as variable-angle epifluorescence microscopy (VAEM), and it is shown how to distinguish the two techniques in practical microscopy. These TIRF images show the highest signal-to-background ratio, and it is demonstrated that they can be used for single-molecule microscopy. Rare protein events, which would otherwise be masked by the average molecular behaviour, can therefore be detected, including the conformations and oligomerization states of interacting proteins and signalling networks in vivo. The demonstration of the application of TIRFM and single-molecule analysis to plant cells therefore opens up a new range of possibilities for plant cell imaging.

Stamen development is governed by a conserved genetic pathway, within which the role of hormones has been the subject of considerable recent research. Our understanding of the involvement of gibberellin (GA) signalling in this developmental process is further advanced than for the other phytohormones, and here we review recent experimental results in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) that have provided insight into the timing and mechanisms of GA regulation of stamen development, identifying the tapetum and developing pollen as major targets. GA signalling governs both tapetum secretory functions and entry into programmed cell death via the GAMYB class of transcription factor, the targets of which integrate with the established genetic framework for the regulation of tapetum function at multiple hierarchical levels., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In higher plants, timely degradation of tapetal cells, the innermost sporophytic cells of the anther wall layer, is a prerequisite for the development of viable pollen grains. However, relatively little is known about the mechanism underlying programmed tapetal cell development and degradation. Here, we report a key regulator in monocot rice (Oryza sativa), PERSISTANT TAPETAL CELL1 (PTC1), which controls programmed tapetal development and functional pollen formation. The evolutionary significance of PTC1 was revealed by partial genetic complementation of the homologous mutation MALE STERILITY1 (MS1) in the dicot Arabidopsis (Arabidopsis thaliana). PTC1 encodes a PHD-finger (for plant homeodomain) protein, which is expressed specifically in tapetal cells and microspores during anther development in stages 8 and 9, when the wild-type tapetal cells initiate a typical apoptosis-like cell death. Even though ptc1 mutants show phenotypic similarity to ms1 in a lack of tapetal DNA fragmentation, delayed tapetal degeneration, as well as abnormal pollen wall formation and aborted microspore development, the ptc1 mutant displays a previously unreported phenotype of uncontrolled tapetal proliferation and subsequent commencement of necrosis-like tapetal death. Microarray analysis indicated that 2,417 tapetum- and microspore-expressed genes, which are principally associated with tapetal development, degeneration, and pollen wall formation, had changed expression in ptc1 anthers. Moreover, the regulatory role of PTC1 in anther development was revealed by comparison with MS1 and other rice anther developmental regulators. These findings suggest a diversified and conserved switch of PTC1/MS1 in regulating programmed male reproductive development in both dicots and monocots, which provides new insights in plant anther development.

Controlling male fertility is an important goal for plant reproduction and selective breeding. Hybrid vigour results in superior growth rates and increased yields of hybrids compared with inbred lines; however, hybrid generation is costly and time consuming. A better understanding of anther development and pollen release will provide effective mechanisms for the control of male fertility and for hybrid generation. Male sterility is associated not only with the lack of viable pollen, but also with the failure of pollen release. In such instances a failure of anther dehiscence has the advantage that viable pollen is produced, which can be used for subsequent rescue of fertility. Anther dehiscence is a multistage process involving localized cellular differentiation and degeneration, combined with changes to the structure and water status of the anther to facilitate complete opening and pollen release. After microspore release the anther endothecium undergoes expansion and deposition of ligno-cellulosic secondary thickening. The septum separating the two locules is then enzymatically lysed and undergoes a programmed cell death-like breakdown. The stomium subsequently splits as a consequence of the stresses associated with pollen swelling and anther dehydration. The physical constraints imposed by the thickening in the endothecium limit expansion, placing additional stress on the anther, so as it dehydrates it opens and the pollen is released. Jasmonic acid has been shown to be a critical signal for dehiscence, although other hormones, particularly auxin, are also involved. The key regulators and physical constraints of anther dehiscence are discussed.

Background: ChIP-Seq, which combines chromatin immunoprecipitation (ChIP) with high-throughput massively parallel sequencing, is increasingly being used for identification of protein-DNA interactions in vivo in the genome. However, to maximize the effectiveness of data analysis of such sequences requires the development of new algorithms that are able to accurately predict DNA-protein binding sites., Results: Here, we present SIPeS (Site Identification from Paired-end Sequencing), a novel algorithm for precise identification of binding sites from short reads generated by paired-end solexa ChIP-Seq technology. In this paper we used ChIP-Seq data from the Arabidopsis basic helix-loop-helix transcription factor ABORTED MICROSPORES (AMS), which is expressed within the anther during pollen development, the results show that SIPeS has better resolution for binding site identification compared to two existing ChIP-Seq peak detection algorithms, Cisgenome and MACS., Conclusions: When compared to Cisgenome and MACS, SIPeS shows better resolution for binding site discovery. Moreover, SIPeS is designed to calculate the mappable genome length accurately with the fragment length based on the paired-end reads. Dynamic baselines are also employed to effectively discriminate closely adjacent binding sites, for effective binding sites discovery, which is of particular value when working with high-density genomes.

The Arabidopsis thaliana ABORTED MICROSPORES (AMS) gene encodes a basic helix-loop-helix (bHLH) transcription factor that is required for tapetal cell development and postmeiotic microspore formation. However, the regulatory role of AMS in anther and pollen development has not been fully defined. Here, we show by microarray analysis that the expression of 549 anther-expressed genes was altered in ams buds and that these genes are associated with tapetal function and pollen wall formation. We demonstrate that AMS has the ability to bind in vitro to DNA containing a 6-bp consensus motif, CANNTG. Moreover, 13 genes involved in transportation of lipids, oligopeptides, and ions, fatty acid synthesis and metabolism, flavonol accumulation, substrate oxidation, methyl-modification, and pectin dynamics were identified as direct targets of AMS by chromatin immunoprecipitation. The functional importance of the AMS regulatory pathway was further demonstrated by analysis of an insertional mutant of one of these downstream AMS targets, an ABC transporter, White-Brown Complex homolog, which fails to undergo pollen development and is male sterile. Yeast two-hybrid screens and pull-down assays revealed that AMS has the ability to interact with two bHLH proteins (AtbHLH089 and AtbHLH091) and the ATA20 protein. These results provide insight into the regulatory role of the AMS network during anther development.

Background: SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction., Methodology/principal Findings: A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2., Conclusions/significance: The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.

Poaceae, one of the largest flowering plant families in angiosperms, evolved distinct inflorescence and flower morphology diverging from eudicots and other monocots. However, the mechanism underlying the specification of flower morphology in grasses remains unclear. Here we show that floral zygomorphy along the lemma-palea axis in rice (Oryza sativa) is partially or indirectly determined by the CYCLOIDEA (CYC)-like homolog RETARDED PALEA1 (REP1), which regulates palea identity and development. The REP1 gene is only expressed in palea primordium during early flower development, but during later floral stages is radially dispersed in stamens and the vascular bundles of the lemma and palea. The development of palea is significantly retarded in the rep1 mutant and its palea has five vascular bundles, which is similar to the vascular pattern of the wild-type lemma. Furthermore, ectopic expression of REP1 caused the asymmetrical overdifferentiation of the palea cells, altering their floral asymmetry. This work therefore extends the function of the TCP gene family members in defining the diversification of floral morphology in grasses and suggests that a common conserved mechanism controlling floral zygomorphy by CYC-like genes exists in both eudicots and the grasses.

The control of male fertility is of vital importance for crop breeding, hybrid generation, and the control of pollen release. Recent development in the analysis of Arabidopsis male sterile mutants has meant that there is a greater understanding of the gene regulatory networks controlling maternal development of the anther and the resultant sporophytes. With the advent of the genome sequence and tools to allow the analysis of gene function, this knowledge base is now extending into the monocot crop rice. This has shown high levels of similarity between the networks of pollen development in Arabidopsis and rice, which will serve as valuable tools to understand and manipulate this developmental pathway further in plants.

Background: Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule., Results: We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods., Conclusion: The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene is critical for viable pollen formation and has homology to the PHD-finger class of transcription factors; however, its role in pollen development has not been fully defined. We show that MS1 transcription appears to be autoregulated by the wild-type MS1 transcript or protein. Using a functional green fluorescent protein (GFP) fusion to analyze the temporal and spatial expression of MS1, we demonstrate that the MS1:GFP protein is nuclear localized within the tapetum and is expressed in a developmentally regulated manner between late tetraspore and microspore release, then rapidly breaks down, probably by ubiquitin-dependent proteolysis. Absence of MS1 expression results in changes in tapetal secretion and exine structure. Microarray analysis has shown that 260 (228 downregulated and 32 upreglated) genes have altered expression in young ms1 buds. These genes are primarily associated with pollen wall and coat formation; however, a number of transcription factors and Cys proteases have also been identified as the putative primary regulatory targets of MS1. Ectopic expression of MS1 alters transcriptional regulation of vegetative gene expression, resulting in stunted plants with increased levels of branching, partially fertile flowers and an apparent increase in wall material on mature pollen. MS1 therefore plays a critical role in the induction of pollen wall and pollen coat materials in the tapetum and, ultimately, the production of viable pollen.