The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.charophytes ͉ land plants ͉ Chara ͉ Coleochaete ͉ Closterium D iversity in form, a hallmark of extant species, is probably caused by modifications of ancestral gene networks regulating development and by the generation of novel developmental processes (1). The evolution of transcription factors, which have critical functions in development, through gene duplication and subsequent functional divergence has been hypothesized to be a major force in developmental evolution (reviewed in ref.2). The adaptation of green plants to a terrestrial environment and their subsequent diversification are tightly linked to the evolution of the body plan of land plants (3, 4). Members of the MADS-box gene family regulate various aspects of development in flowering plants and therefore were probably involved in the evolution of the morphology of land plants.MADS-box genes are characterized by the conserved MADS domain and are found in a wide range of eukaryotes including metazoans, fungi, slime mold, and green plants (5). These genes have been classified into several groups (6). MIKC C -and MIKC*-type MADS-box genes contain intervening (I), keratinlike (K), and C-terminal (C) domains (7,8). They are present in all major land plant taxa including seed plants, pteridophytes, and bryophytes but have not been found in other organisms such as green algae (5, 6, 9). The Arabidopsis genome (10) contains Ϸ38 MIKC C -type and 5 MIKC*-type genes (6, 9). The MIKC*-type genes tend to form a monophyletic group, including several subgroups of genes with unknown functions (9), but the MIKC Ctype MADS-box genes have been classified into about a dozen subfamilies with diverse functions and expression patterns (reviewed in ref. 6). The floral homeotic genes ar...

HIDENORI URATA, YOICHI TANABE, AKIRA KJYONAGA, MASAHARU IKEDA, HIROAKI TANAKA, MUNEH1RO SHINDO, AND KLKUO ARAKAWA SUMMARY After a general clinical observation period of over 4 weeks, 20 essential hypertensive subjects (Japanese) were randomly divided into two groups. One group (n = 10; 4 men and 6 women; 51.4 ± 2.8 years of age) agreed to physical training using bicycle ergometer exercise with the intensity at blood lactate threshold for 60 minutes three times a week for 10 weeks, while the other group (n = 10; 4 men and 6 women; 51.0 ± 2.9 years of age) did no particular physical training and was followed once a week as the control. Changes in blood pressure, hemodynamics, and humoral factors of the exercised group were compared with values in the controls. The following significant changes were found only in the exercised group. Blood pressure was significantly (p<0.01) reduced. Whole blood and plasma volume indices were significantly reduced (p<0.05, p<0.01, respectively). The change in ratio of serum sodium to potassium positively correlated with the change in systolic blood pressure (r = 0.76, p<0.02). Plasma norepinephrine concentrations both at rest and at the workload of blood lactate threshold during graded exercise tests were significantly reduced (p<0.05, p<0.02 respectively) after 10 weeks of exercise training. The change in the resting level of plasma norepinephrine positively correlated with that in the mean blood pressure. No such changes were observed in the control group. In both groups, body weight and urinary sodium excretion showed no statistically significant changes. In conclusion, the antihypertensive effect of mild exercise training was confirmed in a well-matched, controlled study, and reduction in blood volume and plasma norepinephrine concentrations had an apparent association. (Hypertension 9: 245-252, 1987) KEY WORDS • blood pressure • training • electrolytes • catecholamines • lactate D ESPITE widespread interest in nonpharmaco logical therapy for hypertension, physical exercise has not gained acceptance for its antihypertensive effectiveness and mechanisms. 12 This may be attributed to the fact that there has been no well-controlled study of factors such as age, sex, race, number, and life-style in matched hypertensive control subjects. Even when positive effects 1 " 8 were obtained, the related mechanisms remained unclear. Supported in part by giants-in-aid from the Japanese Ministries of Education, Science, and Culture (B-58480246), Health and Welfare (60A-6), Uehara Memorial Foundation, and from the Fukuoka University Research Institute.Address for reprints: Kikuo Arakawa, M.D., The Department of Internal Medicine, Fukuoka University, 7-45-1 Nanakuma, Fukuoka, Japan 814-01.Received February 4, 1986; accepted October 31, 1986. We reported the antihypertensive effect of exercise training with the workload at blood lactate threshold and noted a significant reduction of plasma catecholamine concentration and an elevation of plasma prostaglandin E concentration. 9 ' l0 In addit...

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Coordination of metal ions significantly contributes to protein structures and functions. Here we constructed a fusion protein, consisting of a de novo designed, metal-ion-binding, trimeric coiled-coil and a circularly permutated green fluorescent protein (cpGFP), where the fluorescent emission from cpGFP was induced by metal ion coordination to the coiled-coil. A circularly permutated GFP, (191)cpGFP(190), was constructed by connecting the original N- and C-termini of GFP(UV) by a GGSGG linker and cleaving it between Asp(190) and Gly(191). The metal-ion-binding coiled-coil, IZ-HH, was designed to have three alpha-helical structures, with 12 His residues in the hydrophobic core of the coiled-coil structure. IZ-HH exhibited an unfolded structure, whereas it formed the trimeric coiled-coil structure in the presence of divalent metal ions, such as Cu(2+), Ni(2+), or Zn(2+). The fusion protein (191)cpGFP(190)-IZ-HH was constructed, in which (191)cpGFP(190) was inserted between the second and third alpha-helices of IZ-HH. Escherichia coli cells, expressing (191)cpGFP(190)-IZ-HH, exhibited strong fluorescence when the Cu(2+) and Zn(2+) ions were present in the medium, indicating that they passed through the cell membrane and induced the proper folding of the (191)cpGFP(190) domain. This strategy, in which protein function is regulated by a metal-ion-responsive coiled-coil, should be applicable to the design of various metal-ion-responsive, nonnatural proteins that work both in vitro and in vivo.

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.

Thirty-one Japanese with essential hypertension were divided into training (n = 21) and non-training (n = 10) groups. Physical training of 10 weeks was instituted after 4 or more weeks of observation, and changes in blood pressure and serum concentrations of taurine and other amino acids were investigated. The workload in physical training was predetermined by the submaximal multistage graded exercise test on a bicycle ergometer, and the blood lactate threshold which reflects approximately 40-60% of maximal oxygen uptake was chosen. The hypertensive patients underwent bicycle ergometer training for 60 minutes, three times a week for 10 weeks. Blood pressures were significantly decreased by 14.8/6.6 mmHg in systole/diastole in the training group, but not in the non-training group. Serum concentrations of taurine and cystine were increased significantly by 26% and 287%, in the training group. Increase in serum asparagine (11%), histidine (6%) and lysine (7%) concentrations was also significant, only in the training group. Plasma norepinephrine level and whole blood and plasma volumes were significantly reduced. The change in serum taurine level was significantly negatively correlated with the change in plasma norepinephrine. In addition there was a significant positive correlation between the change in plasma norepinephrine and the change in diastolic blood pressure in the training group. Based on these results, the increase in serum taurine which is known for its antihypertensive activity could contribute, at least in part through the reduction in plasma norepinephrine level, to the antihypertensive effect of exercise.

Recent progress in plant molecular genetics has revealed that floral organ development is regulated by several homeotic selector genes, most of which belong to the MADS-box gene family. Here we report on SrMADS1 , a MIKC c -type MADS-box gene from Selaginella , a spikemoss belonging to the lycophytes. SrMADS1 phylogenetically forms a monophyletic clade with genes of the LAMB2 group, which are MIKC c genes of the clubmoss Lycopodium , and is expressed in whole sporophytic tissues except roots and rhizophores. Our results and the previous report on Lycopodium MIKC c genes suggest that the ancestral MIKC c gene of primitive dichotomous plants in the early Devonian was involved in the development of basic sporophytic tissues such as shoot, stem, and sporangium.

The desmid Closterium peracerosum-strigosum-littorale complex, which is the closest unicellular sister to land plants, is the best characterized of the charophycean green algae with respect to the process of sexual reproduction. To elucidate the molecular mechanism of intercellular communication during sexual reproduction, we created a normalized cDNA library from mixed cells of the sexual and the vegetative phases and generated a cDNA microarray. In total, 3,236 expressed sequence tags, which were classified into 1,615 nonredundant groups, were generated for cDNA microarray construction. Candidate genes for key factors involved in fertilization, such as those that encode putative receptor-like protein kinase, leucine-rich-repeat receptorlike protein, and sex pheromone homologs, were up-regulated during sexual reproduction and/or by the addition of the purified sex pheromones, and the expression patterns of these genes were confirmed by quantitative real-time polymerase chain reaction analysis. This first transcriptome profile of Closterium will provide critical clues as to the mechanism and evolution of intercellular communication between the egg and sperm cells of land plants.