Structure and Expression of a Heat-Shock Protein 83 Gene of Pharbitis nil

Four cDNA clones representing mRNAs whose levels were affected by a photoperiod that induces flowering in Pharbitis nil were isolated by a differential hybridization screening procedure. The level of mRNAs represented by three clones (12L, 15L, and 17L) increased following a photoperiod that induces flowering and that represented by the fourth clone (clone 27) increased under conditions in which flowering was inhibited. DNA sequence analysis showed that one cDNA, clone 17L, is homologous to members of the 83- to 90-kD heat-shock protein (hsp) gene family. The corresponding gene, hsp83A, was isolated and its DNA sequence was determined. hsp83A encodes a protein that exhibits 70% amino acid identity with Drosophila melanogaster HSP83. The P. nil hsp83A gene contains two introns within the coding region. hsp83A mRNA was not detectable in cotyledons of plants grown in continuous light, but its level increased transiently following a 14-h dark period and reached a maximum 2 h after the lights were turned on. A dramatic increase in the level of hsp83A mRNA was also found 2 h after an end-of-day dark treatment. Genomic Southern blot analysis demonstrated that the P. nil hsp83-90 gene family consists of at least six members, one of which appears to be constitutively expressed in the light.     

Promoter Activity of Phenylalanine Ammonia-Lyase Gene of Pharbitis nil in Arabidopsis

Promoter activity of phenylalanine ammonia-lyase (PAL) gene of Pharbitis nil was examined by introducing a PAL:GUS construct into Arabidopsis. GUS staining was observed in vascular bundles of hypocotyl and cotyledons, endodermal cells of the primary root, hydathodes, stigma and pollens of mature flower, abscission zones of petals and sepals and inner layer of seed coat. Light induced GUS expression in cotyledons and the upper part of hypocotyl in which anthocyanin was accumulated. Wounding also induced GUS expression. Endogenous PAL activity increased earlier than the GUS activity directed by the PAL promoter.     

Rhythmicity of Flowering in Pharbitis nil

When young seedlings of Pharbitis nil are grown under continuous light, except for a single inductive dark period, they flower to a varying degree, depending on when this dark period is given. Plants become sensitive to this induction approximately three days after the seedlings emerge from the soil. The expression of flowering varies in a rhythmic fashion for three or more cycles, when an inductive dark period is given at progressively later times. The time between maximum expression of flowering is 24 hours or somewhat longer. It appears necessary that the inductive dark period be of sufficient duration, to only partially induce the plants to flower for this rhythm to be expressed. Under the conditions employed in this study, this duration is 12 hours. If this rhythm is endogenous, it exists at least from when the plants emerged from the soil since no environmental cues are given after that time, and it raises questions of the interpretations of data from previous studies with this organism.     

Physical Basis of Flower-opening in Pharbitis nil

Flower buds of Pharbitis nil cut from plants growing in thefield open rapidly when subjected to darkness (20–25°C)or low temperature (20°C) in light. Petals of the buds arethe sites of photo- and thermo-perception; flower-opening iscaused mainly by the epinasty of petal midribs. ¹Dedicated to Professor Dr. Erwin Bunning on the occasion ofhis 75th birthday. (Received October 23, 1980; Accepted December 15, 1980)     

Gibberellins in Immature Seeds of Pharbitis nil

Two new gibberellins, gibberellins A₂₆ and A₂₇ were isolated from immature seeds of Japanese morning-glory (Pharbitis nil) and their structures were elucidated as I and IX.     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

Carbon Dioxide and Flowering in Pharbitis nil Choisy

The effects of photoperiod on floral and vegetative development of Pharbitis nil were modified by atmospheric CO₂ concentrations maintained during plant growth. Short day (SD) photoperiods caused rapid flowering in Pharbitis plants growing in 0.03 or 0.1% CO₂, while plants in long day (LD) conditions remained vegetative. At 1 or 5% CO₂, however, flower buds were developed under both the SD and LD photoperiods. Flowering was earliest in the plants exposed to SD at low CO₂ concentrations which formed floral buds at stem node 3 or 4. At high CO₂ concentrations, floral buds did not form until stem node 6 or 7. Both high CO₂ concentrations and LD photoperiods tended to enhance stem elongation and leaf formation.     

Calmodulin from Pharbitis nil: Purification and Characterization

A protein, identifiable as calmodulin (CaM), has been isolated from the seedling tissue of Pharbitis nil. The method has been developed to isolate a high quality protein from plant tissue containing the high content of polyphenols. This protein was relatively heat-stable and bound to hydrophobic resin in calcium-dependent manner. It was recognized by the antibody against pea and carrot, but did not bind to antibody against Dictyostelium discoideum. This protein had M_r of 15 kDa and 18.5 kDa in the presence and absence of Ca₂₊, respectively, and was able to stimulate calmodulin-deficient cAMP phosphodiesterase. Based on its migration on SDS-PAGE gels, M_r and binding to anti-CaM antibodies it was deduced that calmodulin from P. nil is essentially identical to calmodulin isolated from other plants.     

Gibberellins in Immature Seeds of Pharbitis nil

A new gibberellin, gibberellin A₂₀ (GA₂₀), was isolated from immature seeds of morning-glory (Pharbitis nil). Its structure was established as 4aα, 7α-dihydroxy-1β-methyl-8-methylenegibbane-1α, 10β-dicarboxylic acid-1→4a lactone (I) on the basis of its physicochemical analysis as well as chemical evidences. GA₂₀ shows marked growth promoting activities on dwarf maize d₂ and d₅ but weak activities on d₁, rice seedling and dwarf pea.     

Phytochrome in Norflurazon-treated seedlings of Pharbitis nil

The properties of phytochrome have been measured by dual-wavelength spectropho-tometry in the cotyledons of the short-day plant Pharbitis nil Choisy cv. Violet, where it is known to play a role in flower induction. In plants de-etiolated by a single white light period (4 h or longer), destruction of the far-red absorbing form of phytochrome (P_fr) was twice as rapid as after 10 min red light. A small fraction of P_fr was stable. After de-etiolation by a period of white light (6 h or longer) the rapid decrease of P_fr during the first 30 min was accompanied by a rapid increase of the red absorbing form of phytochrome (P_fr). This rapid increase of P_fr is probably due to dark reversion. Long term synthesis of phytochrome was inhibited by the presence of P_fr. Phytochrome synthesised in darkness showed the etiolated-plant type characteristics and underwent rapid destruction upon photoconversion to P_fr. The stable P_fr identified here is possibly that pool of phytochrome associated with the long term promotive process in flower induction, and the rapidly reverting P_fr is that pool associated with the night break inhibition of flowering.     

裂叶牵牛psah基因的克隆,序列分析及其表达研究

从裂叶牵牛（Ｐｈａｒｂｉｔｉｓ ｎｉｌ Ｃｈｏｉｓｙ）子叶的ｃＤＮＡ文库中,分离了一个５２０ ｂｐ 的ｐｓａＨｃＤＮＡ 克隆,ＤＮＡ序列分析表明其开放阅读框架由１４４ 个氨基酸构成,包括４９ 个氨基酸长的转运肽和９５ 个氨基酸长的成熟ＰＳⅠＨ（ｓｕｂｕｎｉｔ Ｈｏｆｐｈｏｔｏｓｙｓｔｅｍ Ⅰ）。利用Ｎｏｒｔｈｅｒｎ 杂交技术,发现在高等植物中ｐｓａＨ 基因的表达明显地受内生昼夜节奏的调控,在幼苗阶段呈现出组织特异性,并证明光是迅速启动ＰＮｐｓａＨ基因高水平表达的重要因子     

Chloride absorption and distribution in chlorine-deficient Pharbitis nil

Determination and distribution of radioactive chloride in Pharbitis nil was investigated by a bio-imaging analyzer. When leaves that contained various amounts of ³⁶Cl were analyzed with the imaging analyzer and then each sample was homogenized and its radioactivity measured in a liquid scintillation counter, radioactive levels recorded by the analyzer were directly proportional to the radioactivity determined by the counter. When plants that had been grown in full nutrient solution were incubated in [³⁶Cl]-containing solution, more activity was found in young leaves than in mature leaves, while little radioactivity was detected in shrivelled leaves and the nonsymptomatic cusp of young leaves of plants that had been grown in chlorine-deficient solution.     

Phytochrome in Pharbitis nil during and after de-etiolation

Phytochrome (P) was measured by in vivo spectrophotometry in the cotyledons of Pharbitis nil Choisy cv. Violet. In etiolated plants exposure to red light (R) results in a rapid fall in P content by destruction of the far-red absorbing form of phytochrome (Pfr). In continuous R or white light the P content falls as a result of destruction to a stable 3% of that originally present. In Norflurazon-treated white light de-etiolated plants, Pfr undergoes reversion in darkness to the red absorbing form of phytochrome (Pr) with a half life of 4 h at 19°C, while total P remains constant. Synthesis of Pr after de-etiolation occurs at a slow rate and is enhanced by terminating the de-etiolation light treatment with far-red light. The results are discussed in relation to the P control of flowering.     

Effects of Light and Temperature on Flower-opening of Pharbitis nil

Flower buds of Pharbitis nil cut from plants growing in thefield opened rapidly when kept in darkness for 8 hr followedby continuous light at 20–25°C, but those kept indarkness for 4 hr opened promptly oniy when the temperatureduring the following light period was kept at 23°C or lower.Buds exposed to continuous light at 25°C did not open, butthose exposed to continuous light at 23°C opened slowly.At a lower temperature, the buds opened rapidly even in continuouslight. When the buds were placed in darkness at 25°C at13:30, 17:30 and 21:30 (artificial light from 17:30 to 21:30),they opened about 10 hr after the onset of darkness regardlessof the time of the onset of darkness, but when the buds werekept at 20°C in light from 13:30, 17:30 and 21:30, theyopened at 3:30–5:30 regardless of the time of transferto the lower temperature. The biological clock which controlsthe time of flower-opening is suggested to be easily reset bya light-off signal, but not by a shift from a normal to lowertemperature (20°C). At the lower temperature, the time offlower-opening probably is determined by the time of the latestpreceding light-off (or light-on) signal. ¹Dedicated to Professor Dr. Erwin Biinning on the occasion ofhis 75th birthday. (Received October 23, 1980; Accepted December 15, 1980)     

Biochemical evidence for a cGMP-regulated protein kinase in Pharbitis nil

It is known that the level of cGMP is modulated in response to a number of stimuli in plant cells but intracellular events distal to cGMP metabolism are not clear. Cyclic GMP-dependent protein kinase (Pk-G) is a major effector of cGMP action in animals and yeasts. We wanted to determine whether such kinase is present in plant cells. A soluble protein kinase was isolated from seedlings of Pharbitis nil and purified following purification methods including anion-exchange and affinity-chromatography. The enzyme consists of a single polypeptide of M(r) 70 kDa as determined by SDS-PAGE. From conventional modulators only cyclic GMP, when applied in low concentration, was able to accelerate the enzyme activity in the presence of histones. The enzyme autophosphorylated on serine and threonine residues and phosphorylated some substrates only on serine residues. Mixture of histones and histones H2B, H3 were the best phosphate acceptors. The process of autophosphorylation was accelerated by a low concentration of cGMP and reduced by high concentration of this second messenger. Antibodies raised against catalytic domain of animals Pk-G I alpha and beta cross-reacted with protein kinase from Pharbitis nil tissue. These data, taken together, demonstrate the presence of functional enzyme, which activity is regulated by cGMP and allow to classify this protein kinase as a member of the second messenger regulated group of enzymes.     

Engineering of a Polydisperse Small Heat-Shock Protein Reveals Conserved Motifs of Oligomer Plasticity

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