Gene expression in the soybean seed axis during germination and early seedling growth

Copy-DNA clones have been obtained that distinguish eight messenger mRNAs, moderately abundant in the axes of the germinating soybean (Glycine max (L.) Merr.) seedling. These clones have been used to characterize the size of the mRNAs and to anlyze the accumulation of the mRNAs at different time points and in different parts of the axis during germination and early seedling growth. Three of the mRNAs accumulate to a substantial level by 9 h, a time point before either the beginning of growth or the accumulation of polyribosomes. Four other mRNAs reach a substantial level only at 24 h, a period when rapid seedling growth is occurring. Those mRNAs whose accumulation begins at 24 h were found only in the top (hypocotyl) half of the 24-h seedlings, while the remaining mRNAs were present also in the bottom half of the seedlings in different amounts. By 44 h, the bottom 0.5 cm of the seedlings, i.e., the region of meristematic growth, had little or none of the mRNAs, with the exception of one mRNA. These temporal and spatial observations indicate that many of the mRNAs are not involved simply in the general maintenance of ongoing cell proliferation, but that they may be related to differentiation during early seedling formation. Further, the early accumulating mRNAs may be functioning in regulating the onset of seedling growth.Abbreviations cDNA copy DNA - poly(A)⁺RNA polyadenylated RNA     

Developmental expression and perturbation of arabinogalactan-proteins during seed germination and seedling growth in tomato

Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins present throughout the plant kingdom. A synthetic chemical reagent, ( β - d -Gal)₃ Yariv reagent, specifically binds AGPs and can be used for histochemical staining, isolating and probing the function of AGPs. Here, the role of AGPs in tomato ( Lycopersicon esculentum Mill. cv. UC82B) seed germination and seedling growth was examined by following expression of AGPs during these events and by treatment with ( β - d -Gal)₃ Yariv to perturb AGP function. AGP expression changed during germination and seedling development both quantitatively and qualitatively as revealed by analysis of total AGP content, crossed electrophoresis patterns, RNA blots using LeAGP-1 probe, and western blots with LeAGP-1, JIM13, and MAC207 antibodies. ( β - d -Gal)₃ Yariv treatment of seeds and developing seedlings did not affect percent seed germination, but markedly inhibited seedling growth in roots and to a lesser degree in shoots. Root growth inhibition encompassed reductions in overall root length, epidermal root cell elongation, root cell numbers and root hair formation. This growth inhibition was reversible following removal of ( β - d -Gal)₃ Yariv. In a related experiment, water uptake by tomato seedlings was greatly inhibited by ( β - d -Gal)₃ Yariv treatment. Based on these experiments, AGPs are clearly associated with tomato seedling development and likely to function in root growth, more specifically in cell elongation, cell proliferation, root hair formation and water uptake.     

Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

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.     

Developmental regulation of a Rhizobium cell surface antigen during growth of pea root nodules.

A monoclonal antibody, AFRC MAC 203, was used to examine the expression of a nodule-induced cell surface antigen associated with lipopolysaccharide in Rhizobium leguminosarum bv. viciae 3841. Silver-enhanced immunogold-labeled tissue sections revealed that, in very young tissues of pea root nodules, the nodule-induced form of lipopolysaccharide antigen was not expressed either by rhizobia in the infection thread or by bacteria recently released into the plant cell cytoplasm. In the more mature regions of the nodule, the antigen was expressed by membrane-enclosed bacteroids, including immature forms that had not yet expressed the enzyme nitrogenase and were not yet Y shaped. Immunogold labeling of thin sections revealed that the MAC 203 antigen, but not the nitrogenase, was also expressed by bacteria in infection threads situated in and between bacteroid-containing plant cells in mature nodule tissue.     

Mapping salinity tolerance during Arabidopsis thaliana germination and seedling growth

To characterize and dissect genetic variation for salinity tolerance, we assessed variation in salinity tolerance during germination and seedling growth for a worldwide sample of Arabidopsis thaliana accessions. By combining QTL mapping, association mapping and expression data, we identified genomic regions involved in salinity response. Among the worldwide sample, we found germination ability within a moderately saline environment (150 mM NaCl) varied considerable, from >90% among the most tolerant lines to complete inability to germinate among the most susceptible. Our results also demonstrated wide variation in salinity tolerance within A. thaliana RIL populations and identified multiple genomic regions that contribute to this variation. These regions contain known candidate genes, but at least four of the regions contain loci not yet associated with salinity tolerance response phenotypes. Our observations suggest A. thaliana natural variation may be an underutilized resource for investigating salinity stress response.     

Biochemical changes during germination and seedling growth in Cuscuta campestris

Changes in the contents of starch, protein, DNA, RNA, total phosphorus, acid soluble phosphorus and inorganic phosphorus, and in the activities of some enzymes of carbohydrate, amino acid, nucleic acid and phosphate metabolism were studied during the germination of Cuscuta campestris seeds. The results are expressed on per seed basis.
Starch content in Cuscuta seeds showed a steady decline with most of it depleted by the end of the eighth day of germination. Protein content increased with germination up to 48 h and then decreased. RNA and DNA contents increased to a maximal level on the fourth day of germination and then decreased. Total phosphorus in the seeds remained almost unchanged during the period of study. Both trichloroacetic acid soluble and inorganic phosphorus increased until the third day and then decreased. Phytin was rapidly hydrolyzed with little being detectable by the seventh day of germination. Glucose-6-phosphate dehydrogenase increased with germination, while fructose bisphosphate aldolase which is indispensable for glycolysis, decreased with germination. Ribonuclease and deoxyribonuclease increased till the third and fourth day, respectively, and then decreased. Aspartate and alanine aminotransferases showed a maximum on the second day and then decreased. Activities of alkaline fructose-1,6-bisphosphatase and phytase were absent in the dry seeds and appeared only on the second day of germination. Both α- and β-amylase activities were present in the dry seed.     

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase

 Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination. Received: 28 September 1999 / Accepted: 27 December 1999     

Photomodulation of strigolactone biosynthesis and accumulation during sunflower seedling growth

Present investigations report the presence of strigolactones (SLs) and photomodulation of their biosynthesis in sunflower seedlings (roots, cotyledons and first pair of leaves) during early phase of seedling development. Qualitative analyses and characterization by HPLC, ESI-MS and FT-IR revealed the presence of more than one type of SLs. Orobanchyl acetate was detected both in roots and leaves. Five-deoxystrigol, sorgolactone and orobanchol were exclusively detected in seedling roots. Sorgomol was detectable only in leaves. HPLC eluted fraction from seedling roots and leaves co-chromatographing with GR24 (a synthetic SL) could also bring about germination in Orobanche cernua (a weed) seeds, which are established to exhibit SL – mediated germination, thereby indicating the SL identity of the eluates using this bioassay. SLs accumulation was always more in the roots of light-grown seedlings, it being maximum at 4 d stage. Although significant activity of carotenoid cleavage dioxygenase (CCD, the enzyme critical for SL biosynthesis) was detected in 2 d old seedling roots, SLs remained undetectable in cotyledons at all stages of development and also in the roots of 2 d old light and dark-grown seedlings. Roots of light-grown seedlings showed maximum CCD activity during early (2 d) stage of development, thereby confirming photomodulation of enzyme activity. These observations indicate the migration of a probable light-sensitized signaling molecule (yet to be identified) or a SL precursor from light exposed aerial parts to the seedling roots maintained in dark. Thus, a photomodulation and migration of SL precursor/s is evident from the present work.     

Stimulation of celery seed germination and seedling growth with combined ethephon,gibberellin and polyethylene glycol seed treatments

The upper-temperature limit for germination of cv. Celebrity celery seeds incubated in darkness in laboratory tests was slightly raised following a seed soak with a mixture of the gibberellins A₄ and A₇ + ethephon (G + E) but more so by osmotic priming with polyethylene glycol (PEG). However, the best effects were obtained with a combination of growth regulator and priming treatments. Incorporation of growth regulators into the PEG was more effective than soaking seeds in G + E solutions either before or after osmotic priming. In glasshouse tests, seedling emergence was more rapid, percentage emergence was higher and seedling weight was greater from seeds treated with PEG either alone or in combination with G + E than from untreated seeds, water-soaked seeds or seeds given G + E soaks before or after PEG treatment.     

Nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi

Summary In Gibberella fujikuroi, ammonium (NH₄ ⁺) interfered with the production of gibberellic acid (GA₃). Optimal production occurred at 19 mm (NH₄)₂SO₄ and the synthesis of GA₃ was reduced threefold in a medium with 38 mm (NH₄)₂SO₄. Using a resting cell system with mycelia previously grown on two concentrations (19 mm and 38 mm) of (NH₄)₂SO₄, it was found that NH₄ ⁺ depressed synthesis of the gibberellin-synthesizing enzymes. Furthermore, addition of NH₄ ⁺ to a producing system shut off gibberellin formation, indicating that the negative effect of NH₄ ⁺ ions is also due to inhibition of one or more enzymes in the gibberellin biosynthesis pathway. The onset of gibberellin biosynthesis in media with high (38 mm) and low (19 mm) concentrations of (NH₄)₂SO₄ was studied by addition of cycloheximide to batch cultures of various ages. Offprint requests to: B. Brückner     

Developmental regulation of catecholamine levels during sea urchin embryo morphogenesis

Results of a number of pharmacological studies suggest that catecholamines play a regulatory role in cleavage, morphogenesis and cell differentiation during early animal embryonic development. Few studies, however, have actually assayed for levels of catecholamines in these early embryos by methods that are both sensitive and specific. In this investigation the catecholamines dopamine, norepinephrine and epinephrine and their precursor, dopa and metabolites were determined in eight different embryonic stages of the sea urchin, Lytechinus pictus from hatched blastula to late pluteus larva, using high performance liquid chromatography with electrochemical detection. Levels of each of the catecholamines exhibited unique developmental profiles and are consistent with a role for epinephrine in blastula and early gastrula embryos and for norepinephrine in gastrulation. Changes in levels of catecholamine precursor and metabolites suggest a changing pattern of synthetic and metabolic enzyme activity, which can, for the most part, explain the fluctuations in catecholamine levels during development from blastula to the pluteus larva stage.     

Acyl-CoA dehydrogenase activity in pea cotyledon tissue during germination and initial growth

Acyl-CoA dehydrogenase activity has been measured in homogenates of post-imbibition to 14-day-old hydroponically grown pea seeds at daily intervals, using C(4), C(12) and C(16) acyl-CoA substrates. The activity peaks of the different chain-length acyl-CoA dehydrogenases did not transpose at all points and the ratios of the chain-length activities were not constant. It therefore has to be concluded that more than one dehydrogenase is present in pea mitochondria. There was a post-imbibition initial surge of activity with short- and mid-chain-length substrates. The C(16)-handling enzyme first peaked at 3-4 days, which coincided with the onset of plumule unfurling and greening. Further peaks were observed with all three substrates, coinciding with secondary root formation and leaf enlargement and later with cotyledon degeneration. Overall activity showed that the long-chain acyl-CoA dehydrogenase was much more active than the short-chain acyl-CoA dehydrogenase.     

Mobilisation of storage reserves during germination and early seedling growth of sugar beet

The principal storage reserve of sugar beet seeds is starch, which is localised in the perisperm. Additional storage reserves include the seed proteins, albumins, globulins and glutclins, which are exclusively located in the embryo. Soluble sugars are also detectable in all the organs of the mature seed. The time-course of reserve mobilisation in the different organs of the sugar beet ( Beta vulgaris L. cv. Regina) seed during germination and early seedling growth is documented, with particular reference to changes in (a) activities of hydrolases: a-amylase, β-amylase, and α-glucosidases; (b) levels of carbohydrates and (c) proteins. Amylase activities increase substantially in both cotyledons, as well as the perisperm, whereas the increase in α-glucosidase activities is largely confined to the perisperm.     

Thermoperiodic stem elongation involves transcriptional regulation of gibberellin deactivation in pea

The physiological basis of thermoperiodic stem elongation is as yet poorly understood. Thermoperiodic control of gibberellin (GA) metabolism has been suggested as an underlying mechanism. We have investigated the influence of different day and night temperature combinations on GA levels, and diurnal steady-state expression of genes involved in GA biosynthesis (LS, LH, NA, PSGA20ox1, and PsGA3ox1) and GA deactivation (PsGA2ox1 and PsGA2ox2), and related this to diurnal stem elongation in pea (Pisum sativum L. cv Torsdag). The plants were grown under a 12-h light period with an average temperature of 17 degrees C. A day temperature/night temperature combination of 13 degrees C/21 degrees C reduced stem elongation after 12 d by 30% as compared to 21 degrees C/13 degrees C. This was correlated with a 55% reduction of GA1. Although plant height correlated with GA1 content, there was no correlation between diurnal growth rhythms and GA1 content. NA, PsGA20ox1, and PsGA2ox2 showed diurnal rhythms of expression. PsGA2ox2 was up-regulated in 13 degrees C/21 degrees C (compared to 21 degrees C/13 degrees C), at certain time points, by up to 19-fold. Relative to PsGA2ox2, the expression of LS, LH, NA, PSGA20ox1, PsGA3ox1, and PsGA2ox1 was not or only slightly affected by the different temperature treatments. The sln mutant having a nonfunctional PsGA2ox1 gene product showed the same relative stem elongation response to temperature as the wild type. This supports the importance of PsGA2ox2 in mediating thermoperiodic stem elongation responses in pea. We present evidence for an important role of GA catabolism in thermoperiodic effect on stem elongation and conclude that PsGA2ox2 is the main mediator of this effect in pea.     

草木灰对四种松属种子发芽和幼苗生长的影响

火干扰不仅影响森林生态系统的结构与功能,而且影响群落更新与演替。为了研究草木灰对4种松属种子萌芽和幼苗生长的影响,在湖南省株洲市黄丰桥林场采集马尾松、云南松、湿地松和火炬松种子,以马尾松次生林火烧迹地的草木灰为基质,设置对照和3个草木灰处理,进行播种试验及生长观测。结果表明:(1)草木灰处理下4个物种的种子发芽率比对照试验低,并呈现随草木灰含量的增加而降低的趋势,低草木灰含量处理马尾松种子发芽率不及对照一半,中、高草木灰含量处理发芽率更低;云南松、湿地松和火炬松种子发芽率规律类似于马尾松,而高草木灰含量处理发芽率为0。(2)培养皿播种平均发芽时间比花盆播种长,无论草木灰处理还是培养条件(培养皿或花盆),马尾松和云南松种子平均发芽时间比湿地松和火炬松短。(3)4个物种种子播种14周后,对照试验中马尾松和云南松幼苗死亡率最低,而火炬松和湿地松幼苗死亡率相对增加。(4)播种14周后不同试验处理4个物种幼苗生长差异不明显。火炬松、湿地松、云南松和马尾松幼苗的单株平均干重分别达0.038、0.031、0.027、0.024 g。各物种叶干重按降序排列依次是火炬松(0.026 g)湿地松(0.019 g)云南松(0.017 g)马尾松(0.016 g),根和茎的干重比重相当,约占对应物种幼苗叶干重的三分之一。研究表明以草木灰为培养基质,马尾松和云南松种子的发芽率较高,平均发芽时间较短,更适合作为火烧迹地植被恢复先锋树种,为火干扰后选择人工促进天然更新树种提供参考。