Time to rest : Signals in shoot apex developmental transitions underlying dormancy

Vuodenajat rytmittävät monivuotisten kasvien elämää pohjoisella pallonpuoliskolla, jolla varmin merkki lähestyvästä talvikaudesta on asteittain lyhenevä päivänpituus. Kun päivänpituus on lyhentynyt tiettyyn raja-arvoon saakka, kasvu hiipuu ja kasvin kehityksessä tapahtuu suuria muutoksia. Väitöskirjatyössäni tutkittiin mekanismeja, jotka liittyvät pituuskasvun päättymiseen, silmujen lepotilan kehittymiseen ja kärkisilmun muodostumiseen hybridihaavan ja koivuntaimilla lyhyen päivänpituuden seurauksena kasvihuoneolosuhteissa. Vain lepotilaiset silmut selviytyvät luonnossa ankaran talvikauden yli, joten etenkin lepotilan kehittymisen tutkiminen on keskeistä pyrittäessä selvittämään monivuotisille kasveille tyypillisen kasvutavan mekanismeja. Jo pitkään on tiedetty, että täysikasvuiset lehdet vastaanottavat tiedon päivänpituudesta ja lähettävät signaaleja varren johtojänteissä ylöspäin kohti kasvin kärkiosaa. Sen sijaan varren kärjen ja kärkikasvupisteen roolia lepotilan kehittymisessä on selvitetty vain vähän. Kuitenkin juuri kärkikasvupisteen selviytyminen vuodesta toiseen on tärkeää, koska sen jakautumiskykyiset solukot tuottavat kasvin maanpäälliset osat. Tässä työssä tehdyissä varttamiskokeissa osoitettiin, että varren kärki ei ainoastaan vastaanota signaaleja lehdistä ja ajoita toimintaansa niiden mukaan, vaan myös kärjellä itsellään on aktiivinen rooli lepotilan kehittymisessä. Erityisesti kiinnitettiin huomiota kärkikasvupisteen eri alueiden, ns. apikaalimeristeemin ja rib-meristeemin erilaisiin tehtäviin ja pääteltiin, että molemmat vaikuttavat lepotilan kehittymiseen. Kokeissa käytettiin normaalien hybridihaapojen lisäksi siirtogeenisiä hybridihaapoja, jotka eivät lopeta kasvuaan lyhyt päivä –olosuhteissa. Siirtogeeniset hybridihaavat ilmensivät voimakkaasti fytokromi A -nimistä valon vastaanottajamolekyyliä rib-meristeemin alueella, mikä saattoi osaltaan vaikuttaa poikkeavaan pituuskasvukäyttäytymiseen. Myös useiden lepotilan kehittymiseen liittyvien geenien ilmenemisessä havaittiin poikkeavuuksia verrattuna ei-siirtogeenisiin kontrolleihin, joiden silmuissa kehittyi lepotila lyhyt päivä –altistuksen seurauksena. Väitöskirjatyössäni havaittiin, että myös kaasumainen kasvihormoni etyleeni toimii viestinvälittäjänä silmujen lepotilan kehittymisessä ja vaikuttaa etenkin lepotilan oikeaan ajoittumiseen. Etyleenillä huomattiin olevan määräävä rooli päätesilmun muodostumisessa: siirtogeeniset koivut, jotka eivät aisti etyleeniä, eivät muodostaneet päätesilmua. Silti siirtogeeniset koivut vaipuivat lepotilaan, joskin myöhemmin kuin ei-siirtogeeniset kontrollit. Tämän perusteella todettiin, että lepotilan ja päätesilmun kehittyminen ovat erillisiä kehitystapahtumia, vaikka ne saattavatkin ajoittua osaksi päällekkäin.

Studies on photoblastic germination in lettuce seed

It was shown, with the aid of osmotic inhibition of germination, that the action of the far-red-absorbing form of phytochrome (Pf) in promoting germination can be completed even if the seed is held under conditions where germination is not possible. An effect of the continuing action of Pf beyond the point of complete germination promotion was demonstrated by enhancement of germination rate after removal of the osmotically active solute.

Previous reports that the rate of growth in water of seeds freed from the expansion-restricting endosperm is independent of the state of phytochrome were confirmed. However, a marked, phytochrome-mediated enhancement of the growth potential of such seeds was demonstrated through restricting water uptake by incubation in an osmoticum.

An experimental system, utilizing the appearance of a geotropic curvature in the radicle of the excised axial portion of the seed, was developed for more detailed studies of the phytochrome-enhanced growth potential. It was possible to demonstrate the light effect in water as well as in osmotica; this apparently is not possible with de-endospermed entire seeds. As in intact seeds, the effect of the continuing action of Pf is to enhance the rate of the response. Secretion of a chemical inhibitor of growth by the endosperm as a possible mechanism of induction of light sensitivity has been ruled out.

The phytochrome-dependent rate of appearance of geotropic curvature in osmotica is paralleled in time by a similar dependence of the rate of early extension growth of the embryonic axis. Only the first small increment of growth is a differentially responsive to red (R) and far-red (F); the rate of later increase in length is independent of the light regime.

It was shown that the high concentrations of gibberellic acid required for germination promotion in the intact seed are due at least in part to a diffusion barrier in the endosperm, and that the occasional reports in the literature of the ineffectiveness of kinetin are probably due to the same phenomenon. It was shown that gibberellin, like red light, enhances the growth potential of the axis, but kinetin does not. The difference in rates of response obtained after R-irradiation or gibberellin treatment, together with other results reported in the literature, strongly suggests that gibberellic acid and red light promote germination by different means. The idea that kinetin promotes germination by yet another mechanism, probably operating in the cotyledons, was supported through two different experimental approaches.

The phenomenon of temperature-dependent dark germination was examined in detail, using a wide range of both temperatures and incubation times. With the aid of the half-seed system, it was demonstrated that the promotive effect of low temperature on germination could not be due to a low optimum temperature for early growth of the radicle, since the rate of that process increased with increasing temperature, up to the highest temperature used.

It was shown that phytochrome does not function at high temperatures. This fact is of considerable importance in interpreting the phenomenon of thermodormancy, since in the literature only a small part of the effect of high temperature has been ascribed to an effect on phytochrome, and at that, only to an acceleration of dark reversion of Pf to the red-absorbing form of phytochrome (Pr). Partial denaturation of phytochrome may also make some contribution.

It was shown that the germination-promoting effect of low temperature depends on the presence of Pf, and concluded that low temperatures act by delaying or preventing transformation of Pf. Support for the assumption that Pf, not Pr, is the active form of phytochrome in lettuce seeds was drawn from the same evidence.

Attempts to stimulate germination by repeated irradiation with F over relatively prolonged incubation times resulted in failure, as have similar attempts reported in the literature. However, an enhancement of growth potential in the half-seed system by the maintenance of a small amount of Pf over long periods at ordinary temperatures by repeated irradiation with F was demonstrated.

It was observed that cold storage of the dry seed prevents or delays loss of dark dormancy during post-harvest storage. No change in the response of the half-seed in osmoticum to R and F was observed in seeds that has lost dark dormancy; that is, no internal change took place to measurably increase the growth potential of the embryonic axis. This suggests that the endosperm is the seat of changes responsible for after-ripening of photoblastic lettuce seed.

生长素水平以及光敏色素基因表达与水稻生殖生长的关系

第一部分 光敏核不育水稻农垦58S花药IAA的免疫组织化学分析 光周期敏感核不育水稻农垦58S是研究光周期调节花发育作用机理的好材料。在此方面已取得很大进展。有关植物激素的研究已发现长日照诱导农垦58S不育的信号传导环节之一是IAA的亏缺。本文首次应用免疫组织化学分析方法对长日和短日处理后的农垦58S和对照农垦58花药中的IAA进行了定位研究和相对水平的比较。结果表明此方法可反映游离态IAA在花药中的分布及其相对水平的变化。从雌雄蕊原基形成期至单核晚期的五个时期中，经长日照处理的农垦58S花药中的IAA水平都低于短日照处理的农垦58S及在不同光周期处理下的家垦58花药。对花药中生长素的亏缺与育性的关系以及IAA亏缺的原因也进行了讨论。 第二部分 不同水稻品种成花诱导阶段光周期敏感性及光敏色素mRNA丰度的比较 对光敏核不育水稻十多年的研究表明，光敏色素是感受光周期信号调节水稻成花诱导和育性转换的主要光受体。同时还发现光敏核不育基因导入不同遗传背景后，其基因表达条件，如临界光长、光敏温度范围、光温互补作用强弱等都表现明显差异。为了进一步探讨水稻光周期反应的作用机理，了解不同品种遗传背景对光周期信号感受的特点，我们选取籼稻、粳稻、早熟、晚熟共11个品种，比较光照阶段在长同、短同条件下叶片光敏色素B mRNA含量的差异。初步结果表明，光敏色素B的转录不受光调控，在长日、短日处理条件下没有明显差别。在多数品种间光敏色素B表达没有明显差异，说明其与品种感光性、籼粳性无明显相关。只有早熟粳稻铁粳23的光敏色素B mRNA含量较高，是一个例外。实验结果尚需进一步重复。这方面的工作可以为水稻发育光温互作本质的研究积累一些初步资料。

光敏核不育水稻中光敏色素的分析

本文报道了在育性转换敏感期光周期处理对光敏核不育水稻（农垦58S）及农垦58最新全展叶中光敏色素Ⅰ(PhyA)水平的影响PartI）．在10个光周期处理的最后一个暗期结束前,收获每株水稻的最上部二叶。PhyA用酶联免疫吸附测定法(ELISA)测定。 结果表明：0.5%(v／v)聚乙烯亚胺(PEI)可除去水稻叶片粗提液中干扰ELISA的物质;所用的ELISA专一性地检测水稻PhyA。和长日照(LD)处理相比，短日照(SU)处理导致农垦58S中PhyA的相对含量增加38.5%;而农垦58只增加18.5%。显然，在较长的暗期条件下（SD），农垦58S中PhyA的合成比农垦58快。SD处理下大量增加的PhyA可能和农垦58S的育性恢复有关。 上述结果也说明：在同一品种甚至不同品种的植株间，PhyA水平均易受光周期影响而剧烈变化。 为了进一步验证农垦58S中PhyA较快积累的推论，比较了农垦58s和农垦58幼苗（三叶期）在一延长暗期(24h)中PhyA的积累时程。和育性转换敏感期的植株相似，农垦58S幼苗中PhyA积累速度快于农垦58。在暗期开始6h后，这种差异更明显。这一结果证实了过去的假设：甲基化水平低的农垦58PhyA基因可能比农垦58PhyA基因更活跃地表达。 PhyA和PhyB同时存在于水稻叶片中。为了探讨PhyB是否参与农垦58S雄性不育的调节，在育性转换敏感期每日光期结束、暗期开始开始前进行短暂的FR照射实验(即end-of- dayFR irradiations)。EOD FR反应应由PhyB介导。和SD下的对照相比，经过10次EODFR处理(EOD FR+SD)的农垦58S植株抽穗和开花期都相应地推迟2天，而花粉败育率和种子结实率都没有变化。 EODFR处理抑制了农垦58的开花，但花粉育性几乎不受影响。 综上所述，可能是PhyA而不是PhyB参与调节农垦58S的雄性不育。 另外，本文采用免疫印迹(Immunoblotting or Western blotting)比较了农垦58S和农垦58黄化苗(3天龄)中PhyA的相对含量(PartⅡ)。 结果表明，RPA可以专一性地检测两品种中120KD多肽。该肽在照射R或FR后对内源蛋白酶水解的敏感性不同，照射FR后，该肽易降解产生116KD的片段;照射R后，相对较稳定。因此，上述120KD多肽是水稻PhyA。未观察到农垦58S和农垦58的PhyA在免疫原性、分子量及内源蛋白酶解水解带型有差异。定量分析表明农垦58s黄化苗中PhyA的相对含量比农垦58多40%。这一结果和上述光周期处理的结果是相辅相成的。由于干种子、以及吸涨36h以前的水稻胚中均检测不PhyA的存在，因此两品种间PhyA含量的差异是PhyA蛋白重新合成的结果。 活体低温(80K)荧光光谱分析表明：农垦58黄化苗（3天龄）具有典型光敏色素(主要为PhyA）的荧光发射，其最大波长为683.8nm，而农垦58S以及由其转育来的培矮64s都缺少明显的光敏色素峰。显然，农垦58S和农垦58的PhyA荧光光谱特性有所不同。这一差异是否和雄性不育有关仍待深入研究。 本文第三部分比较了农垦58S和农垦58黄化苗（6天龄）最初转到白光下(4h)合成叶绿素的情况。无论是短暂红光(R)处理或对照，农垦58幼苗合成叶绿素的量（在白光下4h）都多于农垦58S。由于R促进叶绿素合成的效果可被随后的远红光照射(FR)逆转，因此水稻幼苗中叶绿素合成是在光敏色素的控制下。FR逆转性在农垦58S中似乎更完全。连续FR（12h最有效）促进叶绿素合成的效果在农垦58S中更明显，但叶绿素合成的量（在白光下4h）仍是农垦58多。然而，对于自然光周期下生长的幼苗（2-4叶期），农垦58S的叶绿素含量明显高于农垦58。文中讨论了这种差异的可能原因。

The Physarum polycephalum Genome Reveals Extensive Use of Prokaryotic Two-Component and Metazoan-Type Tyrosine Kinase Signaling.

Physarum polycephalum is a well-studied microbial eukaryote with unique experimental attributes relative to other experimental model organisms. It has a sophisticated life cycle with several distinct stages including amoebal, flagellated, and plasmodial cells. It is unusual in switching between open and closed mitosis according to specific life-cycle stages. Here we present the analysis of the genome of this enigmatic and important model organism and compare it with closely related species. The genome is littered with simple and complex repeats and the coding regions are frequently interrupted by introns with a mean size of 100 bases. Complemented with extensive transcriptome data, we define approximately 31,000 gene loci, providing unexpected insights into early eukaryote evolution. We describe extensive use of histidine kinase-based two-component systems and tyrosine kinase signaling, the presence of bacterial and plant type photoreceptors (phytochromes, cryptochrome, and phototropin) and of plant-type pentatricopeptide repeat proteins, as well as metabolic pathways, and a cell cycle control system typically found in more complex eukaryotes. Our analysis characterizes P. polycephalum as a prototypical eukaryote with features attributed to the last common ancestor of Amorphea, that is, the Amoebozoa and Opisthokonts. Specifically, the presence of tyrosine kinases in Acanthamoeba and Physarum as representatives of two distantly related subdivisions of Amoebozoa argues against the later emergence of tyrosine kinase signaling in the opisthokont lineage and also against the acquisition by horizontal gene transfer.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Chemotaxonomic relationships in Celastraceae inferred from principal component analysis (PCA) and partial least squares (PLS)

This paper describes a chemotaxonomic analysis of a database of triterpenoid compounds from the Celastraceae family using principal component analysis (PCA). The numbers of occurrences of thirty types of triterpene skeleton in different tribes of the family were used as variables. The study shows that PCA applied to chemical data can contribute to an intrafamilial classification of Celastraceae, once some questionable taxa affinity was observed, from chemotaxonomic inferences about genera and they are in agreement with the phylogeny previously proposed. The inclusion of Hippocrateaceae within Celastraceae is supported by the triterpene chemistry.

MEDIATOR25 acts as an integrative hub for the regulation of jasmonate-responsive gene expression in arabidopsis

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression.

Florescimento em cana-de-açúcar

O florescimento da cana-de-açúcar é um processo fisiológico complexo formado por vários estádios de desenvolvimento, e cada estádio tem a sua própria necessidade ambiental e fisiológica. Os fatores externos relacionados com o florescimento são: fotoperíodo, temperatura, umidade e radiação solar, além da fertilidade do solo. Por outro lado, os fatores internos envolvem fitocromo, hormônios, florígeno, ácidos nucleicos, dentre outros. A intensidade do processo de florescimento e as consequências na qualidade da cana-de-açúcar variam com a variedade e com o clima. A redução do volume de caldo é o principal fator no qual o florescimento interfere, resultante do aumento do teor de fibras. Como as demais Poaceae, a cana-de-açúcar floresce, frutifica e morre, garantindo a perpetuação da espécie. Dessa forma, o homem procura interferir na natureza tentando evitar o florescimento da cana-de-açúcar, seja por meio de melhoramento genético ou por meio de reguladores vegetais. em áreas comerciais de produção de cana-de-açúcar, onde há condições ideais para o florescimento da cultura, é recomendado o uso de variedade com potencial menos florífero. E, quando não é possível esse manejo varietal, o uso de inibidores do florescimento é a melhor alternativa para evitar mais perdas no conteúdo de sacarose.

Efeito da temperatura e da luz na germinação de sementes de Acacia polyphylla DC.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of light quality on germination and initial growth of canephori seedlings Peltophorum dubium (Sprenge) Taub.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of light and temperature on seed germination in guava (Psidium guajava L. Myrtaceae)

The germination of seeds of Psidium guajava L. (Myrtaceae) is controlled by phytochrome. Guava seeds can germinate with at least one hour per day of irradiation of high red:far-red ratio light preceeded or followed by shade light, indicating that phytochrome B controls germination in these conditions. Under alternating temperatures, in a range of at least 5degreesC, seeds will germinate in darkness, suggesting that in gaps of the canopy, when seeds are covered by a thin layer of soil they will germinate once the alternating temperatures are experienced. Under these conditions phytochrome A is responsible for the control of guava seed germination.

Influence of light and temperature on seed germination of Cereus pernambucensis Lemaire (Cactaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of the seed coat in the light sensivity in Raphanus sativus L. cv. redondo gigante seeds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Achene dimorphism in Bidens pilosa L. (Asteraceae) as determined by germination test

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)