The transcription factors BEL1 and SPL are required for cytokinin and auxin signaling during ovule development in Arabidopsis

Hormones, such as auxin and cytokinin, are involved in the complex molecular network that regulates the coordinated development of plant organs. Genes controlling ovule patterning have been identified and studied in detail; however, the roles of auxin and cytokinin in ovule development are largely unknown. Here we show that key cytokinin pathway genes, such as isopentenyltransferase and cytokinin receptors, are expressed during ovule development. Also, in a cre1-12 ahk2-2 ahk3-3 triple mutant with severely reduced cytokinin perception, expression of the auxin efflux facilitator PIN-FORMED 1 (PIN1) was severely reduced. In sporocyteless/nozzle (spl/nzz) mutants, which show a similar phenotype to the cre1-12 ahk2-2 ahk3-3 triple mutant, PIN1 expression is also reduced. Treatment with the exogenous cytokinin N-6-benzylaminopurine also altered both auxin distribution and patterning of the ovule; this process required the homeodomain transcription factor BELL1 (BEL1). Thus, this article shows that cytokinin regulates ovule development through the regulation of PIN1. Furthermore, the transcription factors BEL1 and SPL/NZZ, previously described as key regulators of ovule development, are needed for the auxin and cytokinin signaling pathways for the correct patterning of the ovule

EFFECT OF HIGH TEMPERATURE ON OVULE DEVELOPMENT IN FIELD PEA (Pisum sativum L.)

Field pea (Pisum sativum L.) is a cool-season crop that is highly vulnerable to high temperature especially during flowering. Temperatures exceeding 28℃ cause abortion of flowers and young fruits in the field, leading to severe yield loss of the crop. In this research, I aimed to investigate the impact of high temperature on ovule development during the reproductive development of pea. Assessments of gynoecium, ovule development, ovule viability, seed set, and ovule abortion from several cultivars exhibiting a wide range in heat tolerance revealed that high temperature altered the normal progression of ovule development under both growth chamber and field conditions. Plants with open flowers at the first reproductive node, but with closed mature buds at the second reproductive node, were exposed to high temperature (35℃/18℃ day/night) for 4 days under growth chamber conditions. The gynoecium evaluated at the first four reproductive nodes of these plants showed contrasting effects of high temperature among nodes and cultivars. A larger size of gynoecium components, such as ovary, style, and stigma, was identified at the youngest reproductive nodes (Node 3) on some heat-treated cultivars compared to the controls, which was consistent with older flower stages found at those nodes. Assessments of embryo sac and ovule size on these nodes revealed that greater size and advanced ovule development were the main effects of high temperature on heat-tolerant cultivars. In turn, less advanced ovule development on diverse nodes of the plants appears to be the factor that separates medium and low heat-tolerant cultivars under heat stress, where medium heat-tolerant cultivars showed poor development at one node, and a low heat-tolerant cultivar at two nodes. Importantly, the occurrence of embryo development at its early stages (zygote to globular-stage embryo) was detected in > 90% of these ovules. A different level of embryo development suggested that high temperature compromised early embryo growth at affected nodes. Ovule viability, analyzed by the presence of callose deposition and reactive oxygen species (ROS), revealed that high temperature could disrupt ovule development in more than one way. An increase of callose accumulation found around the vascular bundle region of ovules suggested that high temperature could disrupt assimilate transport to the embryo sac. Moreover, a heavy presence of ROS was detected in the embryo sac, indicating possible oxidative damage of the embryo sac contents in young ovules, specifically in pods at the raceme’s distal position at young nodes (Node 4). Evaluation of abortion in mature pods confirmed a consistent failure of ovules right after fertilization and ovules containing embryos at early stages of embryo development in heat-treated plants. In the field, the assessment of young ovules and mature pods of 18 cultivars showed a more severe effect of high temperature on ovule development. Ovules collected at 4 days after flowering and a few days (2-3) of high temperature (>28℃) in the field displayed poor embryo sac development, embryo sac decline, and endosperm and embryo growth disruption. Similar to growth chamber conditions, > 90% of these young ovules showed embryos at early development (pro-embryo to globular stage). Finally, seed number reduction in the field occurred mainly because of high ovule abortion (20-57% per pod) at various stages of embryo growth (pro-embryo to late cotyledon stage). Cultivars that showed the least ovule abortion were 40-10, Naparnyk, and CDC Golden, whereas cultivars with the greatest ovule abortion were Carneval, CDC Centennial, and MFR043. Overall, these findings demonstrated that high temperature disrupted normal ovule development, specifically when embryo formation was taking place. Although a certain level of accelerated development was observed on some nodes, poor ovule development on other nodes could be related to a conflict of assimilate availability for an embryo in development. The outcomes from this research provide valuable insights that enlighten how high temperatures hinder the success of reproductive development in field pea. These findings can also be used to select and assess more proficient varieties with high yield performance under warmer environments

ABERRANT TESTA SHAPE encodes a KANADI family member, linking polarity determination to separation and growth of Arabidopsis ovule integuments

The Arabidopsis aberrant testa shape (ats) mutant produces a single integument instead of the two integuments seen in wild-type ovules. Cellular anatomy and patterns of marker gene expression indicate that the single integument results from congenital fusion of the two integuments of the wild type. Isolation of the ATS locus showed it to encode a member of the KANADI (KAN) family of putative transcription factors, previously referred to as KAN4. ATS was expressed at the border between the two integuments at the time of their initiation, with expression later confined to the abaxial layer of the inner integument. In an inner no outer (ino) mutant background, where an outer integument does not form, the ats mutation led to amorphous inner integument growth. The kan1 kan2 double mutant exhibits a similar amorphous growth of the outer integument without affecting inner integument growth. We hypothesize that ATS and KAN1/KAN2 play similar roles in the specification of polarity in the inner and outer integuments, respectively, that parallel the known roles of KAN proteins in promoting abaxial identity during leaf development. INO and other members of the YABBY gene family have been hypothesized to have similar parallel roles in outer integument and leaf development. Together, these two hypotheses lead us to propose a model for normal integument growth that also explains the described mutant phenotypes

A study of effects of low temperature stress on seed development and yield in wheat (Triticum aestivam L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Agricultural Science in Seed Technology at Massey University

Temperature affects the growth, development, fertility and yield of cereals. The degree of sterility and subsequent yield reduction caused by extreme temperature stress depends upon the minimum level and duration of the stress temperature applied and the stage of plant development at the time of stress. An experiment was conducted in which three low temperature regimes (-4°, -2°C and +3°C) were applied at 5 different stages of plant growth (from 1 day before anthesis to 9 days after anthesis) for a period of 6 hours with pre- and post- conditioning periods of 6 and 4 hours respectively. The results showed that the minimum temperature reached determined the nature and severity of temperature injury in Karamu wheat. Complete floret sterility was evident when a -4°C temperature was imposed at the pre-anthesis or anthesis stages of plant development; florets in any position of the head being equally affected. A -4°C temperature stress applied 3 days after anthesis produced 50% and 5% seed formation in primary and secondary heads, respectively. This seed formation mainly occurred in the basal florets of the apical and central spikelets of the head, however the seeds formed did not develop after stress and subsequent viable seed yield was zero. At the later stages, 6 or 9 days after anthesis a -4°C temperature stress had no significant effect on seed numbers. However there was a substantial negative effect on seed development and viability so that subsequent viable seed yield was zero. Temperature stresses of +3°C and -2°C had no significant effects on seed formation, development and viable seed yield when stresses were applied at any of the stages of plant development tested. The percentage of seed formation was highest in the two basal florets of the central and apical portions of the head compared to that in the two basal florets of the bottom of the head and to the distal florets of all spikelets. The percentage sterility in terms of relative sterility (percentage 'D + R' type ovules) and sterility index (percentage 'D' type ovules) was also described. It was found that in 'Karamu' wheat 16% to 33% rudimentary florets were a common feature, such structures included tiny basal, sterile spikelets and the terminal florets of all spikelets. Morphological and anatomical differences in ovules harvested at different stages of development from different treatments were observed. Ovules were classified into 6 groups for assessment of seed development. (A = apparently not fertilised, B = swollen and conical shaped, C = developing, D = shrivelled and shrunken, E = shrunken with reduced conical shape, R = rudimentary). Possible pathways to seed formation and development can be estimated from the data. A probable pathway to normal seed development is A to B to C. However, in the case of unsuccessful seed formation and development, the pathway is likely to be A to D,A to B to D or A to B to C to D. Further detailed electron microscope work is needed to enable a complete description and understanding of the pathways of seed development in stressed and unstressed plants. Such knowledge is needed to provide a logical basis for the development of cultivars with increased cold tolerance, fertility and yield

Ovule development: genetic trends and evolutionary considerations

Much of our current understanding of ovule development in flowering pants is derived from genetic and molecular studies performed on Arabidopsis thaliana. Arabidopsis has bitegmic, anatropous ovules, representing both the most common and the putative ancestral state among angiosperms. These studies show that key genetic determinants that act to control morphogenesis during ovule development also play roles in vegetative organ formation, consistent with Goethe’s “everything is a leaf” concept. Additionally, the existence of a common set of genetic factors that underlie laminar growth in angiosperms fits well with hypotheses of homology between integuments and leaves. Utilizing Arabidopsis as a reference, researchers are now investigating taxa with varied ovule morphologies to uncover common and diverged mechanisms of ovule development

In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

Background: MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results: We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion: This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses

Development and germination of Sandersonia aurantiaca (Hook.) seeds : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Biology and Biotechnology at Massey University

Sandersonia aurantiaca (Hook.) has recently become an important horticultural crop through its economic value for export of its cut flowers and tubers. Little information however is available on seed structure, morphology, development and propagation. The main objectives of this study were to investigate the pattern of seed development, to find satisfactory methods of improving the seed germination and to assess possible mechanisms of seed dormancy of Sandersonia aurantiaca (Hook.). Seed development was investigated by fixing plant material in FAA solution, embedding in paraffin, and staining with safranin-fast green. A series of sections were examined and photographed under a microscope. Both embryo and endosperm development in Sandersonia show close similarity to development in Allium fistulosum (Alliaceae). Embryo development passes through early globular, late globular, elongated spheroidal and linear embryo development stages. Endosperm development conforms to the Nuclear type. Freely-growing walls between the endosperm nuclei may be associated with the embryo sac wall as projections. The structure of the mature seeds is very similar to that of Iris (Iridaceae) seeds. The small, linear embryo is embedded in the endosperm which constitutes most of the seed volume. Such small, linear embryos may be one reason for embryo dormancy in Sandersonia seed. A special structure (a conical or cylindrical protuberance) is observed in the inner part of the seed coat, which may combine with a lignified layer (and perhaps including the endosperm) to contribute to the coat-imposed domancy in this species. Eighty five treatments were firstly used to improve the germination percentage of Sandersonia seed. Only the treatment in which seeds scarified firstly with sandpaper for 1 min and then nicked near the radicle end showed increased germination from 0 to 10.6% by 30 days, at 20°C. Based on this result, 31 new treatment methods were designed in germination experiment 2. Water uptake patterns, allelopathic effect on lettuce seeds and embryo rescue of Sandersonia seed were also studied for assessing the possible mechanisms of dormancy. The findings of the present study suggest that the Sandersonia seeds have double dormancy. The dormancy mechanism is located in both the seed coat and the embryo and it consists of at least two steps that must be activated in sequence before germination can occur. The first step can be activated prematurely by scarifying and nicking the seeds, thus allowing the seed coat to become permeable to water, oxygen or to reduced mechanical restriction. The second step can be activated directly GA3 which stimulates embryo growth. This germination-promoting technique has great potential for Sandersonia for improvement of the germination percentage of seeds from 0 to about 70%, but development on a commercial scale needs further studies

Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B. decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants

Temporal and spatial expression of genes involved in DNA methylation during reproductive development of sexual and apomictic Eragrostis curvula

Recent reports in model plant species have highlighted a role for DNA methylation pathways in the regulation of the somatic-to-reproductive transition in the ovule, suggesting that apomixis (asexual reproduction through seeds) likely relies on RdDM downregulation. Our aim was therefore to explore this hypothesis by characterizing genes involved in DNA methylation in the apomictic grass Eragrostis curvula. We explored floral transcriptomes to identify homologs of three candidate genes, for which mutations in Arabidopsis and maize mimic apomixis (AtAGO9/ZmAGO104, AtCMT3/ZmDMT102/ZmDMT105, and AtDDM1/ZmCHR106), and compared both their spatial and temporal expression patterns during reproduction in sexual and apomictic genotypes. Quantitative expression analyses revealed contrasting expression patterns for the three genes in apomictic vs sexual plants. In situ hybridization corroborated these results for two candidates, EcAGO104 and EcDMT102, and revealed an unexpected ectopic pattern for the AGO gene during germ line differentiation in apomicts. Although our data partially support previous results obtained in sexual plant models, they suggest that rather than an RdDM breakdown in the ovule, altered localization of AtAGO9/ZmAGO104 expression is required for achieving diplospory in E. curvula. The differences in the RdDM machinery acquired during plant evolution might have promoted the emergence of the numerous apomictic paths observed in plants.Fil: Selva, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Siena, Lorena Adelina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Rodrigo, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Garbus, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Ciencias de la Salud; ArgentinaFil: Zappacosta, Diego Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Romero, José Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; ArgentinaFil: Ortiz, Juan Pablo Amelio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Pessino, Silvina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Investigaciones en Ciencias Agrarias de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias. Instituto de Investigaciones en Ciencias Agrarias de Rosario; ArgentinaFil: Leblanc, O.. Universidad de Montpellier; AlemaniaFil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida. Universidad Nacional del Sur. Centro de Recursos Naturales Renovables de la Zona Semiárida; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; Argentin