Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.     

Osmoconditioning prevents the onset of microtubular cytoskeleton and activation of cell cycle and is detrimental for germination of Jatropha curcas L. seeds

• Jatropha curcas is an oilseed crop renowned for its tolerance to a diverse range of environmental stresses. In Brazil, this species is grown in semiarid regions where crop establishment requires a better understanding of the mechanisms underlying appropriate seed, seedling and plant behaviour under water restriction conditions. In this context, the objective of this study was to investigate the physiological and cytological profiles of J. curcas seeds in response to imbibition in water (control) and in polyethylene glycol solution (osmoticum).
• Seed germinability and reactivation of cell cycle events were assessed by means of different germination parameters and immunohistochemical detection of tubulin and microtubules, i.e. tubulin accumulation and microtubular cytoskeleton configurations in water imbibed seeds (control) and in seeds imbibed in the osmoticum.
• Immunohistochemical analysis revealed increasing accumulation of tubulin and appearance of microtubular cytoskeleton in seed embryo radicles imbibed in water from 48 h onwards. Mitotic microtubules were only visible in seeds imbibed in water, after radicle protrusion, as an indication of cell cycle reactivation and cell proliferation, with subsequent root development. Imbibition in osmoticum prevented accumulation of microtubules, i.e. activation of cell cycle, therefore germination could not be resumed.
• Osmoconditioned seeds were able to survive re‐drying and could resume germination after re‐imbibition in water, however, with lower germination performance, possibly due to acquisition of secondary dormancy. This study provides important insights into understanding of the physiological aspects of J. curcas seed germination in response to water restriction conditions.

     

Proteomics of Arabidopsis seed germination. A comparative study of wild-type and gibberellin-deficient seeds

We examined the role of gibberellins (GAs) in germination of Arabidopsis seeds by a proteomic approach. For that purpose, we used two systems. The first system consisted of seeds of the GA-deficient ga1 mutant, and the second corresponded to wild-type seeds incubated in paclobutrazol, a specific GA biosynthesis inhibitor. With both systems, radicle protrusion was strictly dependent on exogenous GAs. The proteomic analysis indicated that GAs do not participate in many processes involved in germination sensu stricto (prior to radicle protrusion), as, for example, the initial mobilization of seed protein and lipid reserves. Out of 46 protein changes detected during germination sensu stricto (1 d of incubation on water), only one, corresponding to the cytoskeleton component alpha-2,4 tubulin, appeared to depend on the action of GAs. An increase in this protein spot was noted for the wild-type seeds but not for the ga1 seeds incubated for 1 d on water. In contrast, GAs appeared to be involved, directly or indirectly, in controlling the abundance of several proteins associated with radicle protrusion. This is the case for two isoforms of S-adenosyl-methionine (Ado-Met) synthetase, which catalyzes the formation of Ado-Met from Met and ATP. Owing to the housekeeping functions of Ado-Met, this event is presumably required for germination and seedling establishment, and might represent a major metabolic control of seedling establishment. GAs can also play a role in controlling the abundance of a beta-glucosidase, which might be involved in the embryo cell wall loosening needed for cell elongation and radicle extension.     

Expression and inhibition of aquaporins in germinating Arabidopsis seeds

Extensive and kinetically well-defined water exchanges occur during germination of seeds. A putative role for aquaporins in this process was investigated in Arabidopsis. Macro-arrays carrying aquaporin gene-specific tags and antibodies raised against aquaporin subclasses revealed two distinct aquaporin expression programs between dry seeds and young seedlings. High expression levels of a restricted number of tonoplast intrinsic protein (TIP) isoforms (TIP3;1 and/or TIP3;2, and TIP5;1) together with a low expression of all 13 plasma membrane aquaporin (PIP) isoforms was observed in dry and germinating materials. In contrast, prevalent expression of aquaporins of the TIP1, TIP2 and PIP subgroups was induced during seedling establishment. Mercury (5 microM HgCl(2)), a general blocker of aquaporins in various organisms, reduced the speed of seed germination and induced a true delay in maternal seed coat (testa) rupture and radicle emergence, by 8-9 and 25-30 h, respectively. Most importantly, mercury did not alter seed lot homogeneity nor the seed germination developmental sequence, and its effects were largely reversed by addition of 2 mM dithiothreitol, suggesting that these effects were primarily due to oxidation of cell components, possibly aquaporins, without irreversible alteration of cell integrity. Measurements of water uptake in control and mercury-treated seeds suggested that aquaporin functions are not involved in early seed imbibition (phase I) but would rather be associated with a delayed initiation of phase III, i.e. water uptake accompanying expansion and growth of the embryo. A possible role for aquaporins in germinating seeds and more generally in plant tissue growth is discussed.     

Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth

Proteomics of Arabidopsis seeds revealed the differential accumulation during germination of two housekeeping enzymes. The first corresponded to methionine synthase that catalyses the last step in the plant methionine biosynthetic pathway. This protein was present at low level in dry mature seeds, and its level was increased strongly at 1-day imbibition, prior to radicle emergence. Its level was not increased further at 2-day imbibition, coincident with radicle emergence. However, its level in 1-day imbibed seeds strongly decreased upon subsequent drying of the imbibed seeds back to the original water content of the dry mature seeds. The second enzyme corresponded to S -adenosylmethionine synthetase that catalyses the synthesis of S -adenosylmethionine from methionine and ATP. In this case, this enzyme was detected in the form of two isozymes with different p I and M r. Both proteins were absent in dry mature seeds and in 1-day imbibed seeds, but specifically accumulated at the moment of radicle protrusion. Arabidopsis seed germination was strongly delayed in the presence of dl -propargylglycine, a specific inhibitor of methionine synthesis. Furthermore, this compound totally inhibited seedling growth. These phenotypic effects were largely alleviated upon methionine supplementation in the germination medium. The results indicated that methionine synthase and S -adenosylmethionine synthetase are fundamental components controlling metabolism in the transition from a quiescent to a highly active state during seed germination. Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in Arabidopsis only after radicle protrusion.     

Changes in DNA and microtubules during loss and re-establishment of desiccation tolerance in germinating Medicago truncatula seeds

Desiccation tolerance (DT) in orthodox seeds is acquired during seed development and lost upon imbibition/germination, purportedly upon the resumption of DNA synthesis in the radicle cells. In the present study, flow cytometric analyses and visualization of microtubules (MTs) in radicle cells of seedlings of Medicago truncatula showed that up to a radicle length of 2 mm, there is neither DNA synthesis nor cell division, which were first detected in radicles with a length of 3 mm. However, DT started to be lost well before the resumption of DNA synthesis, when germinating seeds were dried back. By applying an osmotic treatment with polyethylene glycol (PEG) before dehydration, it was possible to re-establish DT in seedlings with a radicle up to 2 mm long. Dehydration of seedlings with a 2 mm radicle, with or without PEG treatment, caused disassembly of MTs and appearance of tubulin granules. Subsequent pre-humidification led to an almost complete disappearance of both MTs and tubulin granules. Upon rehydration, neither MTs nor tubulin granules were detected in radicle cells of untreated seedlings, while PEG-treated seedlings were able to reconstitute the microtubular cytoskeleton and continue their normal development. Dehydration of untreated seedlings also led to an apoptotic-like DNA fragmentation in radicle cells, while in PEG-treated seedlings DNA integrity was maintained. The results showed that for different cellular components, desiccation-tolerant seedlings may apply distinct strategies to survive dehydration, either by avoidance or further repair of the damages.     

Two Tomato Expansin Genes Show Divergent Expression and Localization in Embryos during Seed Development and Germination

Expansins are plant proteins that can induce extension of isolated cell walls and are proposed to mediate cell expansion. Three expansin genes were expressed in germinating tomato (Lycopersicon esculentum Mill.) seeds, one of which (LeEXP4) was expressed specifically in the endosperm cap tissue enclosing the radicle tip. The other two genes (LeEXP8 and LeEXP10) were expressed in the embryo and are further characterized here. LeEXP8 mRNA was not detected in developing or mature seeds but accumulated specifically in the radicle cortex during and after germination. In contrast, LeEXP10 mRNA was abundant at an early stage of seed development corresponding to the period of rapid embryo expansion; it then decreased during seed maturation and increased again during germination. When gibberellin-deficient (gib-1) mutant seeds were imbibed in water, LeEXP8 mRNA was not detected, but a low level of LeEXP10 mRNA was present. Expression of both genes increased when gib-1 seeds were imbibed in gibberellin. Abscisic acid did not prevent the initial expression of LeEXP8 and LeEXP10, but mRNA abundance of both genes subsequently decreased during extended incubation. The initial increase in LeEXP8, but not LeEXP10, mRNA accumulation was blocked by low water potential, but LeEXP10 mRNA amounts fell after longer incubation. When seeds were transferred from abscisic acid or low water potential solutions to water, abundance of both LeEXP8 and LeEXP10 mRNAs increased in association with germination. The tissue localization and expression patterns of both LeEXP8 and LeEXP10 suggest developmentally specific roles during embryo and seedling growth.     

Class I chitinase and beta-1,3-glucanase are differentially regulated by wounding,methyl jasmonate,ethylene, and gibberellin in tomato seeds and leaves

Class I chitinase (Chi9) and beta-1,3-glucanase (GluB) genes are expressed in the micropylar endosperm cap of tomato (Lycopersicon esculentum) seeds just before radicle emergence through this tissue to complete germination. In gibberellin (GA)-deficient mutant (gib-1) seeds, expression of Chi9 and GluB mRNA and protein is dependent upon GA. However, as expression occurs relatively late in the germination process, we investigated whether the genes are induced indirectly in response to tissue wounding associated with endosperm cap weakening and radicle protrusion. Wounding and methyl jasmonate (MeJA) induced Chi9 expression, whereas ethylene, abscisic acid, sodium salicylate, fusicoccin, or beta-aminobutyric acid were without effect. Chi9 expression occurred only in the micropylar tissues when seeds were exposed to MeJA or were wounded at the chalazal end of the seed. Expression of Chi9, but not GluB, mRNA was reduced in germinating seeds of the jasmonate-deficient defenseless1 tomato mutant and could be restored by MeJA treatment. Chi9 expression during germination may be associated with "wounding" from cell wall hydrolysis and weakening in the endosperm cap leading to radicle protrusion, and jasmonate is involved in the signaling pathway for this response. Among these treatments and chemicals (other than GA), only MeJA and wounding induced a low level of GluB expression in gib-1 seeds. However, MeJA, wounding, and particularly ethylene induced both genes in leaves, whereas GA induced only Chi9 in leaves. Although normally expressed simultaneously during tomato seed germination, Chi9 and GluB genes are regulated distinctly and tissue specifically by hormones and wounding.     

In Vivo Cell Wall Loosening by Hydroxyl Radicals during Cress Seed Germination and Elongation Growth

Loosening of cell walls is an important developmental process in key stages of the plant life cycle, including seed germination, elongation growth, and fruit ripening. Here, we report direct in vivo evidence for hydroxyl radical (^·OH)-mediated cell wall loosening during plant seed germination and seedling growth. We used electron paramagnetic resonance spectroscopy to show that ^·OH is generated in the cell wall during radicle elongation and weakening of the endosperm of cress (Lepidium sativum; Brassicaceae) seeds. Endosperm weakening precedes radicle emergence, as demonstrated by direct biomechanical measurements. By ³H fingerprinting, we showed that wall polysaccharides are oxidized in vivo by the developmentally regulated action of apoplastic ^·OH in radicles and endosperm caps: the production and action of ^·OH increased during endosperm weakening and radicle elongation and were inhibited by the germination-inhibiting hormone abscisic acid. Both effects were reversed by gibberellin. Distinct and tissue-specific target sites of ^·OH attack on polysaccharides were evident. In vivo ^·OH attack on cell wall polysaccharides were evident not only in germinating seeds but also in elongating maize (Zea mays; Poaceae) seedling coleoptiles. We conclude that plant cell wall loosening by ^·OH is a controlled action of this type of reactive oxygen species.     

黄瓜种子及其萌发期的化感作用研究

以大白菜、萝卜、番茄和黄瓜种子为受体,采用实验室培养皿种子发芽生物测试法研究了黄瓜种子浸提液、种子萌发、胚根和芽苗分泌物、芽苗腐解物和芽苗浸提液的化感效应。结果表明:(1)黄瓜种子浸提液对大白菜、萝卜、番茄和黄瓜种子萌发均有化感抑制作用,即黄瓜种子内含有某些化感抑制物质。(2)在水浸提过的黄瓜种子萌发过程中,它不仅对其近邻套种的大白菜、萝卜和番茄种子萌发产生化感抑制作用,而且其胚根和芽苗分泌物对后茬播种的4种蔬菜种子发芽也表现出不同程度的化感抑制作用;黄瓜芽苗腐解物和芽苗水浸提液也对各受体蔬菜种子发芽与生长产生不同程度的化感抑制作用,且随着腐解芽苗量的增加或浸提液浓度的升高,各受体蔬菜种子的发芽指标值、化感效应指数值和综合效应值随之降低。(3)黄瓜种子浸提液及芽苗各器官的化感物质对黄瓜种子的萌发与生长产生了自毒作用,且黄瓜芽苗腐解物、芽苗浸提液、胚根及芽苗分泌物对受体黄瓜的自毒作用均为最大。研究发现,黄瓜种子浸提液、种子萌发时期以及芽苗各器官的化感物质主要是通过抑制受体胚根的生长而起化感抑制作用,即受体蔬菜种子胚根对化感效应最为敏感;因黄瓜种子及萌发期释放化感物质的途径有所不同,导致受体大白菜、萝卜、黄瓜和番茄的化感响应也不相同;在黄瓜种子萌发和芽苗生长的早期,化感物质即开始在芽苗体内进行合成与积累,一部分可通过胚根和芽苗分泌途径释放到环境中,另一部分可通过芽苗腐解途径释放化感物质,并对受体蔬菜种子萌发与生长表现出较强的化感抑制作用。     

The effects of the phospholipase D-antagonist 1-butanol on seedling development and microtubule organisation in Arabidopsis

The organisation of plant microtubules into distinct arrays during the cell cycle requires interactions with partner proteins. Having recently identified a 90-kDa phospholipase D (PLD) that associates with microtubules and the plasma membrane [Gardiner et al. (2001) Plant Cell 13: 2143], we exposed seeds and young seedlings of Arabidopsis to 1-butanol, a specific inhibitor of PLD-dependent production of the signalling molecule phosphatidic acid (PA). When added to agar growth media, 0.2% 1-butanol strongly inhibited the emergence of the radicle and cotyledons, while 0.4% 1-butanol effectively blocked germination. When normal seedlings were transferred onto media containing 0.2% and 0.4% 1-butanol, the inhibitor retarded root growth by about 40% and 90%, respectively, by reducing cell elongation. Inhibited plants showed significant swelling in the root elongation zone, bulbous or branched root hairs, and modified cotyledon morphology. Confocal immunofluorescence microscopy of root tips revealed that 1-butanol disrupted the organisation of interphase cortical microtubules. Butanol isomers that do not inhibit PLD-dependent PA production, 2- and 3-butanol, had no effect on seed germination, seedling growth, or microtubule organisation. We propose that production of PA by PLD may be required for normal microtubule organisation and hence normal growth in Arabidopsis.     

大金发藓和小蛇苔化学他感作用的生物测定

以绿豆、油菜和萝卜种子为生测材料,以大金发藓（Polytrichum commune Hedw.）和小蛇苔(Conocephalum japonicum(Thunb.) Grolle.)的水提取液为处理液,测定了二种苔藓植物提取液在三种浓度下对三种作物种子萌发率、萌发指数、萌发势、种子活力、幼苗根重和鲜重和根长等指标的影响。发现绿豆是理想的生测材料,种子活力指数、幼苗鲜重、根重和根长是理想的生测指标;二种苔藓植物的水提取液表现出类似于激素样的作用方式,对种子萌发和幼苗生长,表现为低浓度下促进、高浓度时抑制的化学他感效应。     

The effect of cold storing pre-germinated lettuce seeds on radicle development and seedling emergence

The effects on radicle development and seedling emergence of cold storing batches of pre-germinated lettuce seeds for up to 7 days at 0 or 2 °C were investigated in three experiments. Cold storage tended to delay emergence and increase its spread in time. Separate batches of pre-germinated seeds gave different mean radicle lengths and different patterns of emergence and it is likely that small differences in mean temperature during germination were responsible since the length of radicles after germination was linearly related to accumulated day-degrees during the germination period. This suggests that germinating seeds over a period during which a required number of day-degrees is accumulated may produce radicles of the same length for different sowings. The elongation of radicles during cold storage was also linearly related to accumulated day-degrees. At 2 °C it appeared that the longer radicles elongated faster than the smaller ones and this may account for cold storage tending to increase the spread of seedling emergence when the pre-germinated seeds were sown into compost.     

Measurement of the Water Potential of Intact Plant Tissues2PISUM SATIVUM L.)

Through the use of a microthermocouple psychrometer it has becomepossible to measure the water potential over part of the surfaceof a pea seed, starting about 19 hours after sowing in distilledwater, or later in the case of seeds germinating at lower osmoticpotentials, in both instances until well after radicles haveemerged. It has been shown that the potential of air-dry seedsis well below –6,000 joules/kg but increases rapidly duringimbibition, depending upon the water potential of the germinationmedium. Pea seeds subjected to lower external water potentialsgerminate at lower internal water potentials than they exhibitin distilled water. The water potentials of the seeds decreasejust after radicle emergence till the radicle establishes contactwith its germination medium, possibly as a result of demandfor moisture during that period due to incipient cell elongation.No detectable amounts of osmotically active substances are exudedfrom the seed during germination; the pea seed coat restrictsthe entry of polyethylene glycol molecules into the seed untilemergence of the radicle.     

The performance of carrot seeds in relation to their viability

The germination and emergence characteristics, and early seedling growth, of carrot seeds cv. Chantenay red-cored from different sources with a range of germination from 54–94%, was compared. Seeds from protected crops (mean temperature of growth 21°C, r.h. 45–70%) gave higher percentage germination than those from crops grown outdoors (mean temperature 15°C, 70–100% r.h.). Germination was also higher from mature (seed moisture content at harvest 20% or lower) than immature seed (seed moisture content at harvest between 20 and 60%). High percentage germination (>90%) was associated with low mean germination times and low spreads of germination times whilst the reverse was true for low percentage germination. Similar relationships were found for seedling emergence characters in the field although a lower proportion of the viable seeds produced seedlings from slowly than rapidly germinating seed lots. In general, seed lots having a low percentage germination gave greater variability in plant weight than those of higher percentage germination. There was no effect of seed source on radicle or shoot relative growth rates or on post-emergence seedling growth rates.