Maize D4;1 and D5 cyclin proteins in germinating maize. Associated kinase activity and regulation by phytohormones

We have previously reported the expression of four different maize D cyclins during seed germination and showed that cytokinins and auxins stimulate the expression of every cyclin in a differential way. In this paper we characterize the behavior at the protein level of two of these cyclins, CycD5 and CycD4;1. Antibodies were raised against CycD5;2 (which very likely also recognizes D5;1) and CycD4;1 and Western blot studies demonstrated that neither BA nor indol-3 acetic acid (IAA) stimulate cyclin accumulation during germination, compared with control levels. However, phytohormones, particularly IAA, modify the kinase activity associated to D cyclins preferentially at early hours of germination. The associated kinase moiety to D cyclins appears to be of a Cdk-A type because this protein immunoprecipitates with D cyclins and because kinase activity is strongly inhibited by both olomoucine and also by a peptide corresponding to the carboxy end of a maize kip related protein (KRP) protein. There is thus no correlation between mRNA and protein expression for these maize D cyclins during seed germination, although phytohormones may stimulate a signaling cascade that stimulates activation of protein kinase activity in cyclin–Cdk complexes.     

Expression of a maize δ-type DNA polymerase during seed germination

We have cloned a 1563-bp cDNA sequence from a reported 2072-bp cDNA (including a fragment of the 3' UTR region, accession number AY109773 ) corresponding to the carboxy half of maize DNA polymerase δ[ Zea mays delta-type DNA polymerase catalytic subunit (Zmpolδ); EC 2.7.7.7], and its sequence shows an identity of 95, 77 and 74% to rice, soybean and Arabidopsis enzymes, respectively, although identity is even higher if only the polδ-defining domain sequences are considered. An important difference between the monocot and dicot enzymes is the presence in the former of Zn fingers apparently required for binding to the DNA polδ holoenzyme B subunit, which is absent in the dicot enzymes. Expression of Zmpolδ and protein levels during germination remain unchanged; however, polδ shows two peaks of activity, one during early germination, perhaps related to DNA repair processes and a second peak when DNA replication is already in progress, indicating that Zmpolδ is regulated at the post-translational level. Zmpolδ has been found mainly in proliferative tissues in seeds and plantlets, although surprisingly, it is also present in seed endosperm, together with Zm proliferating cell nuclear antigen.     

Plant cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization

The comparative analysis of a large number of plant cyclins of the A/B family has recently revealed that plants possess two distinct B-type groups and three distinct A-type groups of cyclins [1]. Despite earlier uncertainties, this large-scale comparative analysis has allowed an unequivocal definition of plant cyclins into either A or B classes. We present here the most important results obtained in this study, and extend them to the case of plant D-type cyclins, in which three groups are identified. For each of the plant cyclin groups, consensus sequences have been established and a new, rational, plant-wide naming system is proposed in accordance with the guidelines of the Commission on Plant Gene Nomenclature. This nomenclature is based on the animal system indicating cyclin classes by an upper-case roman letter, and distinct groups within these classes by an arabic numeral suffix. The naming of plant cyclin classes is chosen to indicate homology to their closest animal class. The revised nomenclature of all described plant cyclins is presented, with their classification into groups CycA1, CycA2, CycA3, CycB1, CycB2, CycD1, CycD2 and CycD3.     

Proliferating cell nuclear antigen expression in maize seed development and germination: Regulation by phytohormones and its association with putative cell cycle proteins

The proliferating cell nuclear antigen (PCNA) plays a fundamental role in DNA replication and repair and recently, it has been found associated to proteins that control the G1 phase of the cell cycle, such as cyclin D. Maize PCNA cDNA has been cloned and overexpressed in order to raise antibodies. The expression of PCNA has been followed during seed development and seed germination using the homologous antibodies. The protein was found at a constant level during seed development up to 48 days after pollination (DAP) and then the amount declined to very low levels, similar to those found in dry seeds. Upon germination, PCNA levels rose gradually reaching a peak by 20 h germination. Imbibition in the presence of cytokinins (Benzyladenine, BA) produced a sharp increase in amount during the first 3–6 h germination, whereas imbibition in the presence of abscisic acid (ABA) did not alter the pattern of expression as compared with control seeds. Immunoprecipitation experiments showed that PCNA was associated to a putative cyclin D protein during germination and this association was altered by phytohormones. While the complex PCNA-cyclin D-like protein was present along the first 15 h of germination under control conditions, it was dissociated after 6 h if embryo axes germinated in the presence of BA or ABA. However, complex dissociation in the presence of BA was due to degradation of the putative cyclin D protein while in the presence of ABA the putative cyclin D was still present. These results are discussed in the context of seed germination and the cell cycle.     

Modulation of CycD3;1‐CDK complexes by phytohormones and sucrose during maize germination

Maize CycD3;1 associates to CDKA or CDKB1;1 proteins during germination and the complexes formed develop kinase activity. These complexes appear to vary in size as germination proceeds, suggesting association to different sets of proteins. CycD3;1 and associated CDK proteins respond to phytohormones and sucrose. Results revealed a reduction in the CycD3;1 protein amount along germination in the presence of indoleacetic acid (IAA) or abscisic acid (ABA), although in the latter protein levels recover at the end of germination. While the levels of CDKA increase with IAA, they decrease with ABA. Both phytohormones, IAA and ABA, increase levels of CDKB1;1 only during the early germination times. CycD3;1 associated kinase activity is only reduced by both phytohormones towards the end of the germination period. On the other hand, lack of sucrose in the imbibition medium strongly reduces CycD3;1 protein levels without affecting the levels of neither CDKA nor CDKB1;1. The corresponding CycD3;1 associated kinase activity is also severely decreased. The presence of sucrose in the medium appears to stabilize the CycD3;1 protein levels.     

Characterization of a maize beta-amylase cDNA clone and its expression during seed germination.

A maize (Zea mays L.) cDNA clone (pZMB2) encoding beta-amylase was isolated from a cDNA library prepared from the aleurone RNA of germinating kernels. The cDNA encodes a predicted product of 488 amino acids with significant similarity to known beta-amylases from barley (Hordeum vulgare), rye (Secale cereale), and rice (Oryza sativa). Glycine-rich repeats found in the carboxyl terminus of the endosperm-specific beta-amylase of barley and rye are absent from the maize gene product. The N-terminal sequence of the first 20 amino acids of a beta-amylase peptide derived from purified protein is identical to the 5th through 24th amino acids of the predicted cDNA product, indicating the absence of a conventional signal peptide in the maize protein. Recombinant inbred mapping data indicate that the cDNA clone is single-copy gene that maps to chromosome 7L at position 83 centimorgans. Northern blot analysis and in vitro translation-immunoprecipitation data indicate that the maize beta-amylase is synthesized de novo in the aleurone cells but not in the scutellum during seed germination.     

细胞周期蛋白D3在兔妊娠早期子宫和胚胎中的表达与调节

利用免疫组织化学及RT—PCR的方法,研究了细胞周期蛋白D3(Cyclin D3)在兔早期妊娠子宫和着床前胚胎中的表达情况,以揭示CyclinD3在兔胚胎着床过程中的可能作用。结果显示：(1)在兔妊娠第3～8天的子宫近肌层的腺上皮中有CyclinD3免疫染色,并且其表达强度在第7天以后呈现下降的趋势,着床的胚胎中未见CyclinD3免疫染色;(2)在第2～5天的假孕兔子宫的腺上皮中有较强的CyclinD3免疫染色;(3)在发情周期的兔子宫中未见其有表达;(4)在切除卵巢的兔中,注射雌激素后子宫中未见CyclinD3免疫染色,注射孕酮后在子宫腺上皮中有CyclinD3免疫染色,在孕酮和雌激素共同处理后的子宫腺上皮中有较强的CyclinD3免疫染色;(5)利用RT—PCR的方法在早期胚胎中均能检测到CyclinD3,但从胚泡期开始表达上升,到扩展胚泡时其表达最强。上述结果表明,CyclinD3的表达可能对兔胚泡的着床具有一定的调节作用。     

Mitotic cyclin distribution during maize cell division: Implications for the sequence diversity and function of cyclins in plants

Summary Cyclin proteins are components of the regulatory system that controls the orderly progression of the events of cell division. Their sub-cellular location, as well as their fluctuating abundance and their affinities for the cyclin-dependent kinases (CDKs) to which they bind, determine their successive roles during the cell cycle. Here we employ species-specific antibodies to monitor changes in quantity and location of four maize cyclins and maize Cdc2-kinase in dividing maize root tip cells. Maize cyclin Ia occurs in the nuclear matrix and is released when the nuclear envelope breaks down. In contrast, cyclin Ib is cytoplasmic until prophase; it associates transiently with the nuclear envelope and preprophase band (PPB) just before these structures break down and then associates with the condensed chromosomes and spindle region before declining at anaphase. Cyclin II and Cdc2 also occur in the PPB. Occurrence of cyclin Ib and Cdc2 at the PPB concurrent with initiation of breakdown is consistent with previous studies in which microinjection of cyclin-dependent protein kinase indicated that removal of the PPB at the time of nuclear-envelope breakdown is catalysed by a CDK. While cyclins Ia and III are predominantly nuclear prior to mitosis, cyclins Ib and II are predominantly cytoplasmic until prophase then become nuclear. The initial cytoplasmic retention of cyclins Ib and II correlates with their possession of a sequence similar to the cytoplasmic-retention signal of animal cyclin B1. Cyclin II binds to all microtubule arrays during the cell cycle, becoming markedly concentrated in the phragmoplast, and cyclin III associates with the spindle and then the phragmoplast. Cdc2 also occurs in the phragmoplast. Persistence of mitotic cyclins and CDK after mitosis into the cytokinetic stage, as seen in maize, is not paralleled in animal cells, where the cytokinetic mid-body is not so labelled, presumably reflecting the key role of the phragmoplast apparatus in plant cell division.Abbreviations CDK cyclin-dependent kinase - CRS cytoplasmicretention signal - NE nuclear envelope - NEB nuclear-envelope breakdown - NLS nuclear-location signal - PPB preprophase band - FITC fluorescein isothiocyanate - TRITC tetramethylrhodamine isothiocyanate     

外源糖浸种缓解盐胁迫下玉米种子萌发

以玉米品种‘垦玉6号’为材料,在150 mmol·L^-1NaCl胁迫条件下,研究葡萄糖(Glc)和蔗糖(Suc)浸种对玉米种子萌发阶段耐盐性的影响.结果表明: 盐胁迫下,0.5 mmol·L^-1 Glc、Suc浸种可促进玉米种子萌发及幼苗早期生长,其中Glc浸种玉米胚芽和胚根长及相应干质量增加到盐处理的1.5、1.3、2.1、1.8倍;Suc浸种玉米分别增加到1.7、1.3、2.7、1.9倍;盐胁迫下Glc、Suc浸种可减少胚芽中硫代巴比妥酸反应物(TBARS)和过氧化氢(H2O2)含量,与盐处理相比分别降低24.9%、20.6%;Glc、Suc浸种可显著提高盐胁迫下玉米胚芽超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、谷胱甘肽过氧化物酶(GPX)、谷胱甘肽还原酶(GR)的活性,并诱导葡萄糖6 磷酸脱氢酶(G6PDH)活性的升高,其中Glc浸种玉米SOD、APX、GPX、GR、G6PDH活性较盐处理分别提高66.2%、62.9%、32.0%、38.5%、50.5%,Suc浸种玉米较盐处理分别提高67.5%、59.8%、30.0%、38.5%、50.4%;Glc、Suc浸种胚芽中抗坏血酸 (ASA)、谷胱甘肽(GSH)含量及ASA/DHA、GSH/GSSG显著提高,其中G6PDH活性与外源糖诱导的较强的抗氧化能力密切相关.Glc、Suc浸种还可提高盐胁迫下玉米胚芽中K⁺/Na⁺,分别为盐处理的2.3、2.4倍.外源 Glc、Suc浸种可通过提高玉米种子抗氧化能力及维持体内K⁺和Na⁺离子平衡缓解盐胁迫对玉米种子萌发的抑制效应.     

Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction

AtOPT promoter-GUS fusions were constructed for six of the nine known, putative oligopeptide transporters (OPTs) in Arabidopsis thaliana and used to examine AtOPT expression at various stages of plant development. AtOPT1, AtOPT3, AtOPT4, AtOPT6 and AtOPT7 were expressed in the embryonic cotyledons prior to root radicle emergence. Except for AtOPT8, which gave weak expression, all AtOPTs were strongly expressed in post-germinative seedlings with strongest expression in vascular tissues of cotyledons and hypocotyls. Preferential expression of AtOPTs in vascular tissues was also observed in cotyledons, leaves, hypocotyls, roots, flowers, siliques, and seed funiculi of seedlings and adult plants. Differential tissue-specific expression was observed for specific AtOPTs. For example, AtOPT1, AtOPT3 and AtOPT8 were uniquely expressed in pollen. Only AtOPT1 was expressed in growing pollen tubes, while only AtOPT6 was observed in ovules. AtOPT8 was transiently expressed in seeds during early stages of embryogenesis. Iron limitation was found to enhance expression of AtOPT3. These data suggest distinct cellular roles for specific AtOPTs including nitrogen mobilization during germination and senescence, pollen tube growth, pollen and ovule development, seed formation and metal transport.     

Acid phosphatase from maize scutellum: Properties as a function of seed germination

Acid phosphatase (optimum pH at 5.2) purified from maize scutellum both in a state of dormancy and during the first 24 hours of seed germination has a M, of ca 65000, contains 6% neutral sugar, maintains its catalytic activity after succinylation of 52 free amino groups per molecule and does not show the apparent movement of optimum pH from 5.4 to 6.7 in the presence of 4 mM fluoride. Kinetic data showed Michaelian behaviour for the enzymatic hydrolysis of PNP-P and an apparent number of Pi bound per molecule equal to one. Our results also suggest that the increased acid phosphatase activity in maize scutellum as a function of seed germination could be the result of modifications in the enzyme molecule.     

Excess boron reduces polyphenol oxidase activities in embryo and endosperm of maize seed during germination

The effects of increasing concentrations of boron (0, 0.1, 1, 10 and 20 mM) as boric acid on the rate of germination and polyphenol oxidase activities in embryo and endosperm tissues of maize seeds (Zea mays L. cv. Arifiye) were studied. The germination percentage of maize seeds was not affected by boron concentrations up to 10 mM, and decreased by 20 mM. Distilled water and lower boron concentrations (0.1 and 1 mM) increased polyphenol oxidase activities at the beginning of germination up to 12 h whereas its excess levels (10 and 20 mM) decreased polyphenol oxidase activities in embryos and endosperm during germination. Polyphenol oxidase activities with o-diphenolic substrates (caffeic acid, catechol and dopa) were found to be higher than with a monophenolic substrat (tyrosine) in both embryos and endosperms. Further, caffeic acid oxidizing polyphenol oxidase was found to show more activity in embryos of the seeds germinating in distilled water when compared to other substrates.