Shortening of microtubule overlap regions defines membrane delivery sites during plant cytokinesis

© The Author(s), 2016. This is the author's version of the work and is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Current Biology 27 (2017): 514-520, doi:10.1016/j.cub.2016.12.043.Different from animal cells that divide by constriction of the cortex inwards, cells of land plants divide by initiating a new cell wall segment from their centre. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands towards the parental cell wall. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly. How vesicle immobilisation and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell plate formation is lacking. Here we show that membranous material for cell plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4 mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins, we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.The work has been financially supported by HFSP grant RGP0026/2011 to MEJ and GG.2018-01-2

Gametophytic development of Brassica napus pollen in vitroenables examination of cytoskeleton and nuclear movements

Isolated microspores and pollen suspension of Brassica napus “Topas” cultured in NLN-13 medium at 18°C follow gametophytic pathway and develop into pollen grains closely resembling pollen formed in planta. This culture system complemented with whole-mount immunocytochemical technology and novel confocal laser scanning optical technique enables detailed studies of male gametophyte including asymmetric division, cytoskeleton, and nuclear movements. Microtubular cytoskeleton configurationally changed in successive stages of pollen development. The most prominent role of microtubules (MTs) was observed just before and during nuclear migration at the early and mid-bi-cellular stage. At the early bi-cellular stage, parallel arrangement of cortical and endoplasmic MTs to the long axis of the generative cell (GC) as well as MTs within GC under the plasmalemma bordering vegetative cell (VC) were responsible for GC lens shape. At the beginning of the GC migration, endoplasmic microtubules (EMTs) of the VC radiated from the nuclear envelope. Most cortical and EMTs of the VC were found near the sporoderm. At the same time, pattern of MTs observed in GC was considerably different. Multiple EMTs of the GC, previously parallel aligned, reorganized, and start to surround GC, forming a basket-like structure. These results suggest that EMTs of GC provoke changes in GC shape, its detachment from the sporoderm, and play an important role in GC migration to the vegetative nucleus (VN). During the process of migration of the GC to the VC, multiple and thick bundles of MTs, radiating from the cytoplasm near GC plasma membrane, arranged perpendicular to the narrow end of the GC and organized into a “comet-tail” form. These GC “tail” MTs became shortened and the generative nucleus (GN) took a ball shape. The dynamic changes of MTs accompanied polarized distribution pattern of mitochondria and endoplasmic reticulum. In order to confirm the role of MTs in pollen development, a “whole-mount” immunodetection technique and confocal laser-scanning microscopy was essential

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled

Impact of agricultural development scenarios on water resources in Bulgaria, Hungary and Romania

1. Background information In order to examine the environmental and economic effect of low-input and organic types of agriculture in Bulgaria, Hungary and Romania, in 1995 EU-Phare programme funded a pilot project on setting up research and education demonstration centres for sustainable agriculture (EU/AR/301/91). The project’s objective was to “test” the feasibility of sustainable types of agriculture under agro-ecological and socio-economic circumstances of the three countries concerned. The project also had to draft a policy paper for conversion to sustainable agriculture in these countries. Education and research on water quality, biodiversity, market potentials and macro-economic feasibility represented a substantial part of the project activities (ended up in May 1999). The results obtained and experienced gained by this project are used as the frame for a wider discussion regarding the impact of agricultural development scenarios on water resources in the Danube countries. 2. Impact of agriculture on water resources in the Danube River Basin Most agricultural operations, such as soil tillage, manuring, grazing, and irrigation, pose a serious threat to water quality. Agriculture is a substantial water user, and often the single biggest sector responsible for the pollution of European waters. In addition, agriculture threatens water habitats. Available data indicate that in many regions of Europe, agriculture alone is responsible for more than 50% of the total nutrient load borne by water (Znaor, 1999; VROM, 1998; IFEN, 1998; OECD, 1997). Many surface and groundwater resources are exploited beyond sustainable levels and agriculture contributes to this problem by drawing off a considerable volume for irrigation purposes. Around 50% of nitrate and phosphate pollution in the Danube River Basin are attributed to agriculture. The "Danube Integrated Environmental Study" (Haskoning, 1994), a research project involving approximately seventy experts from eleven Danube countries concluded that: 1. Agriculture is the major source of pollution by nitrogen (50%), phosphorus (53%) and pesticides, and contributes significantly to metal (Cd, Cu, Zn), bacteria and viruses emissions 2. Wetlands and related eco-systems still account for a substantial nutrient removal capacity with a high economic value 3. Environmental problems related to water pollution are amplified by the consumption patterns of the Danube basin population, particularly the increase of animal protein consumption 4. The damage to the priority functions (drinking water, recreation, fisheries, ecosystem) of ground and surface water in the Danube basin and part of the north-western shelf of the Black Sea is estimated to be about €4 billion per year. The findings of the Haskoning study were recently confirmed by another study (TG-MWRI, 1997). This study, too, found that more than 50% of N and P in the surface water of the Danube basin derive from agriculture. A study from VITUKI (1997) concluded that a 25% reduction of the nutrient loads (as compared to reference period 1989-91) is required to meet the environmental quality criteria for the Danube River, while a 40% N-reduction and 50% P-reduction scenario would be required for the Black Sea Basin. Experts from the CEE countries have suggested that such reductions (of about 50%) would be feasible if so-called ‘Best Agricultural Practices’ could be generally applied. Obviously both Germany and Austria (Upper Danube) would have to contribute their share in this reduction as well. The above data suggest that agriculture represents by far the most important sector for tacking nutrient pollution in the Danube basin. However, when calculating the amount of water pollution caused by agriculture, the pollution caused by agricultural practices are only part of the problem. Significant levels of pollution are caused by the agri-chemical industry that serves agriculture. It is a very substantial source of pollution. For instance: in Romania a single fertiliser producer contributes 13.4 % to the total Romanian phosphate discharge (60,000 ton) into the Danube river (Toma, 1999). 3. Low-external input agriculture reality and water pollution In Central and Eastern Europe the sustainability issue in agriculture has two additional dimensions as compared to Western Europe. The ‘transition’-process of economic and institutional reform, and the environmental ‘opportunity’ of current low-input reality in agriculture. Due to changes in the price of agricultural commodities and the high price of agricultural inputs, most of the farms in Central and Eastern Europe (CEE) are forced to practice low-input farming. Input levels have fallen to 10-30% of the input levels before 1989. In 1996-98, in most countries the agri-input levels have not increased above 10-50% of the 1989 levels: Bulgaria 10%, Yugoslavia 50%, Poland 50%, Romania 25%, Russia 12% (Kieft, 1999). Even though this is a rather 'forced' agro-social experiment, it is very interesting to study its impact on the agricultural production and water pollution in the Danube River Basin. The various data on inputs, outputs and water pollution show the potential to combine the objectives of agricultural production with environmental protection. Therefore this very reality of low input levels can be perceived also as an environmental opportunity for the region. Especially since a minimum 40-50% nutrients reduction is required to enable ecologically sound functioning of the Black Sea (VITUKI, 1997). This opportunity is only interesting, however, if agricultural production can be increased without increasing pollution again. Studies from some Danube countries (Kieft, 1999), as well as the results of the above-mentioned project indicate that by practising low-external input agriculture, the reduction of yields is not proportional to the reduction of fertiliser and pesticide inputs. Even with relatively low level of agri-chemical input farmers in Bulgaria, Hungary and Romania can achieve interesting output levels, while at the same time reducing or avoiding environmental damages. Maintaining these output levels, however, requires improved farming practices including balanced nutrient management, improved manure handling, erosion control and crop rotations, based on low input techniques. 4. Further agricultural development scenarios and policy options Within the framework of this project, a study measuring the macro-economic impact of sustainable agriculture in Bulgaria, Romania and Hungary was done too (Wit et al. 1999). It showed that large-scale conversion (up to 50% of the arable land) to sustainable agriculture in these countries results in comparable national agricultural production value to that obtained by conventional scenario. The sustainable scenario exhibits even greater economic benefits when the external (environmental) costs accompanying agricultural production are internalised into the price of the produce. By charging a shadow price of ECU1 per kg of nitrogen leached, the sustainable scenario showed even greater economic benefit. It results in net national agricultural production value 16% (Bulgaria), 49% (Hungary) and 5% (Romania) higher than by the conventional scenario. At the same time, the sustainable scenario reduced nitrogen leaching for 34% (Bulgaria), 45% (Hungary) and 35% (Romania) when compared to the conventional scenario. This reduction complies with the targets set up for nutrient reduction for the Danube River and the Black Sea (VITUKI, 1997; Haskonig, 1994). 5. Conclusion Agriculture in Bulgaria, Hungary and Romania, although at a record low input, is not sustainable from an economic or environmental point of view unless it is accompanied by better management practices. A shift to improved low-input agriculture and organic agriculture is beneficial to the national economy and reduces number of environmental and water-related degradations. In order to make this shift possible- a 3-Track policy is proposed: (i) support to pioneering organic farming; (ii) improvement of low-input farming techniques; (iii) conversion of high-input agriculture to integrated agriculture. Establishment of an international facility to support national teams in preparing and implementing this policy would enable a concerted action not only for these three, but also for other Danube countries

Environmental impact and macro-economic feasibility of organic agriculture in the Danube River Basin

Several studies from the mid-nineties pointed to agriculture as the single biggest nutrient polluter of the surface water in the Danube River Basin (Haskoning, 1994). Following these findings, the EU Phare programme awarded in 1995 a four-year education and research project aimed at introducing and assessing feasibility of organic agriculture in three Danube River countries: Bulgaria, Hungary and Romania. So far it is the biggest and most elaborate project on organic agriculture ever implemented in these three countries. The project consisted of a spectrum of training, on-farm demonstration and research/study activities, and prepared strategy papers for policy redesigning. The studies and research performed assessed the impact of organic agriculture on (i) water quality; (ii) biodiversity; (iii) regional markets, and (iv) macro-economic feasibility. In order to comply with the highly demanding project terms of reference and its complexity, an international consortium led by ETC Netherlands (consultancy specialised in sustainable types of agriculture) was formed. The consortium involved partners from Austria, Bulgaria, Hungary, Netherlands, Romania and United Kingdom. This paper is an attempt to summarise the most important findings of the research performed within the framework of this project. However, the diversity of the research topics as well as the limited size of the paper doesn’t allow very elaborated presentation of the results. Therefore this paper outlines only the most essential research findings. RESULTS AND DISCUSSION: Water quality The results obtained show considerable variations between the three countries, as well as among the organic and conventional variants. A true comparison of the country results is not possible, since due to the differences in soil types and research organisation, the three countries were forced to apply different monitoring system. In Hungary, the total amount of seepage water fluctuated over the years. In 1997 there was no significant difference between organic and conventional arable plots, while the organic horticulture plot had some 31% less seepage water than the conventional one. In 1998, the organic arable plot produced 22% less seepage water than the conventional vegetables. However, this was not the case with organic vegetables plot: in 1998 it produced 24% more seepage water than the conventional plot. In 1997 the nitrogen concentration in the soil solution of the organic arable plots was most of the time below 25 mg NO3 l-1, and always below 50 mg NO3 l-1. However, neither organic nor conventional vegetable plots were able to reduce the nitrogen concentration below 60 mg NO3 l-1, and the conventional plot exhibited figures as high as 185 mg NO3 l-1 (sandy soil). In 1998, only the organic arable plot maintained in early spring (and before manuring) the nitrogen concentration below 50 mg NO3 l-1. All other variants exhibited very high nitrogen concentrations, with conventional vegetable plot resulting in figures as high as 265 mg NO3 l-1. The Hungarian results show that the nitrate-N in vegetable plots (both organic and conventional) was much higher than in arable plots, which is due to higher level of nutrients applied, intensive soil cultivation and row cultivation. Contrary to the sandy soil in Hungary, the trial at the loamy soil in Bulgaria gave quite different figures. In the arable plots, organic treatment resulted in nitrate concentrations below 20 mg NO3 l-1 in all soil depths through the year, while the conventional treatment recorded concentrations of 40-60 NO3 l-1. Similar results were obtained from the plots under perennial grassland: the nitrate concentration under organic management was below 20 mg NO3 l-1 throughout the year, while the concentration in the conventional plot amounted to 110 mg NO3 l-1 in spring and 75 mg NO3 l-1 in winter. In Romania the total amount of water transferred from the soil profile to the groundwater (calculated from the difference between cumulated drained water and the cumulated upward flux by capillary rise) was higher in conventional plots than in organic ones. This was due to a lower soil bulk density, a greater water retention and deeper root penetration in the organic plots. A more elaborated information on the results obtained can be found in Hartl and Kromp (1999). Biodiversity assessment The biodiversity was found to be significantly higher (often 100% more) in the organic plots both on weeds and fauna. In Hungary, the organic plots had 19-25 carabid sub species compared to 16-19 found in the conventional plots, while several carabid species occurred significantly more abundant under the organic treatment (Brachinus explodens, Carabus scabriusculus, Dolichus halensis and Harpalus rufipes). Spiders exhibited similar result to that of carabides. In Bulgaria, monitoring of beneficial insects in an orchard (Figure 1), and grain fields found organic treatment to be more favourable than the conventional and integrated ones. The assessment in barley found that earthworms occur significantly more abundant in the organic barley fields. In 1997, one cubic meter of the soil from organic plot contained 41 earthworms, compared to 18 at the conventional plot. In 1998 the difference was even more pronounced, with one cubic meter of organic barley soil harbouring 124 earthworms and conventional only 21. In Romania, the weed survey in maize fields showed a significantly higher biodiversity of weed species in organic maize fields compared to a herbicide-treated conventional one. Further, in Romania organic manure application was found to have a favouring effect on the soil mesofauna. Organic plots exhibited higher densities of total mesofauna as well as of collembolans and oribatid mites compared to the conventional plots. Gammasid mites, occurring only sporadically and with single species in the conventional fields, showed up in higher densities and 2-3 spp. in the organic soils. In Romania, too, the more spiders and Carabus individuals occurred in the organic treatment, as well as a moderately higher diversity of beetle species active on the soil surface. A more elaborated information on the biodiversity results can be found in Hartl and Kromp (1999). Regional marketing study The organic sector represents less than 0.5% of agricultural production in the region. The share of organic market in the Czech Republic is estimated at 0.35%, while in Poland it represents only 0.05% of agricultural production. The organic market stagnates both in Poland and the Czech Republic, while Hungary exports most of its organic production and the market is under expansion. The majority of the organic farms in central and eastern Europe rely on direct marketing, and substantial part of the organic production is still sold through conventional distribution channels. The results obtained suggest that organic farmers in the region can produce for almost the same cost as conventional farmers. The price difference between organic and conventional products seems to be fairly small, with Polish organic market exhibiting somewhat higher differences. The price differences between countries were found to be smaller than differences within a country. The premium price is very small in the countryside, while in big cities consumers sometimes accept considerably higher prices. Cereals and dairy products tend to attract lower premium than other products, particularly fruits and vegetables. More information on the marketing study outcome can be found in Verschuur et al. 1998. Macro-economic feasibility The large-scale conversion study exhibited substantial macro-economic and environmental benefits of the “sustainable scenario”. The calculation suggests that large-scale conversion to the sustainable scenario in Bulgaria, Hungary and Romania would result in gross national agricultural production values comparable to those obtained by the conventional scenario. When the external (environmental) costs of N-leaching are internalised into the price of the produce, by charging a shadow price of 1 EURO per kg of nitrogen leached, the sustainable scenario showed even greater economic benefit. It resulted in higher net national agricultural production values (Romania 5%; Bulgaria 16% and Hungary 40%) than in the conventional scenario. At the same time, the sustainable scenario resulted in substantially lower nitrogen leaching than that in the conventional scenario: 55% in Romania, 66% in Bulgaria and 82% in Hungary. Since the study took into account exclusively the portion of nitrogen lost through leaching, which represents only 39% of the total, the positive environmental impact of organic agriculture migh turn out to be even greater if losses occurring through direct discharge, erosion and run-off were added as well. CONCLUSIONS • The study was too short to establish scientifically reliable results. Only two full seasons were monitored. Therefore the intention of the national research partners to continue this original type of research is laudable. However, the emerging results are in line with comparable research results in western Europe and the USA: in arable farming and pasture the N-leaching is lower under organic as compared to conventional; in horticulture, covering small land areas however, N-leaching tends to be higher in the initial stages of transition as a result of initially high manure dosages. Careful manure management is crucial indeed, both for environmental and for cost-saving reasons. • Preliminary results obtained suggest that the area under organic agriculture generally exhibits positive environmental effects in terms of reduced nutrient leaching, as horticulture covers relatively small percentages of the agricultural land. Leaching is critical on sandy soils under intensive production like in Hungary. In Bulgaria only leaching from organic land use remained below 25 mg NO3 l-1. • Bio-diversity benefits are demonstrated in all 3 countries for various beneficial species and in various crops. In the case of the longer term research in Bulgaria these results are more reliable already and indicate increasing positive effects of organic management on pest- and disease prevention in orchards. • The growing demand for organic food in western Europe could pull the market in central and eastern Europe, on the condition that certification is promoted and larger quantities of produce become available. The Hungarian tactics may be relevant for other countries as well: enhancing organic production via the export market with attractive prices, while creating a local demand in the meantime with slightly lower market prices. • The macro-economic benefits of organic agriculture are very pronounced when the environmental costs of nitrogen leaching are internalised in the production value. This conclusion would justify policy incentives towards environmentally more sustainable agriculture. The emerging results are in line with research results elsewhere. They indicate the potential of organic agriculture to positively contribute to environmental, biodiversity and macro-economic situation in Bulgaria, Hungary and Romania. To enable development of sustainable agriculture in these countries, a 3-track policy is suggested. Its measures should improve environmental and economic performance of the current low-external-input agriculture; promote further development of pioneering organic agriculture; and convert high-external-input regime to integrated agriculture

Depth of dormancy in tomato seeds is related to the progression of the cell cycle prior to its induction

Cell cycle activities are initiated following imbibition of non-dormant seeds. However, it is not known whether cell cycle related events other than DNA replication also remain suppressed in imbibed dormant seeds. The objective of this study was to demonstrate that the transitions between the non-dormant and dormant (both primary and secondary) states are reflected in cell cycle events, such as DNA replication and the changing patterns of the microtubular cytoskeleton involved in the processes of growth and development. The present studies were conducted on seeds from tomato (Lycopersicon esculentum cv. Moneymaker) that possessed primary dormancy or were manipulated to attain secondary dormancy. In addition, a non-dormant abscisic acid (ABA)-deficient mutant, sitw, was used. DNA replication, as measured by flow cytometry, and -tubulin accumulation, analysed by immunoblotting, were compared with immunocytological studies of active DNA synthesis and microtubular cytoskeleton formation. It is shown that the depth of dormancy, which distinguishes primary and secondary dormancy, may depend on the progression of the cell cycle prior to the induction of dormancy