Effects of kinetin and nitrogen on growth rates, pigment and protein contents in wild and phycoerythrin-deficient strains of Hypnea musciformis (Rhodophyta)

Hypnea musciformis (Wulfen in Jacqu.) J.V. Lamour. is the main source for carrageenan production in Brazil and strains with selected characteristics could improve the production of raw material. The effects of kinetin on growth rates, morphology, protein content, and concentrations of pigments (chlorophyll a, phycoerythrin, phycocyanin, and allophycocyanin) were assessed in the wild strain (brown phenotype) and in the phycoerythrin-deficient strain (green phenotype) of H. musciformis. Concentrations of kinetin ranging from 0 to 50 mu M were tested in ASP 12-NTA synthetic medium with 10 mu M nitrate (N-limited) and 100 mu M nitrate (N-saturated). In N-limited condition, kinetin stimulated growth rates of the phycoerythrin-deficient strain and formation of lateral branches in both colour strains. Kinetin stimulated protein biosynthesis in both strains. However, differences between both nitrogen conditions were significant only in the phycoerythrin-deficient strain. In the wild strain, effects of kinetin on concentrations of phycobiliproteins were not significant in both nitrogen conditions, except for chlorophyll content. However, the phycoerythrin-deficient strain showed an opposite response, and kinetin stimulated the phycobiliprotein biosynthesis, with the highest concentrations of phycoerythrin in N-saturated medium, while the highest concentrations of allophycocyanin and phycocyanin were observed in N-limited medium. These results indicate that the effects of kinetin on growth, morphology, protein and phycobiliprotein contents are influenced by nitrogen availability, and the main nitrogen storage pools in phycoerythrin-deficient strain of H. musciformis submitted to N-limited conditions were phycocyanin and allophycocianin, the biosynthesis of which was enhanced by kinetin.

Efficacy of Cosmetic Formulations Containing Dispersion of Liposome with Magnesium Ascorbyl Phosphate, Alpha-Lipoic Acid and Kinetin

The present study aimed to evaluate the photoprotective effects of cosmetic formulations containing a dispersion of liposome with magnesium ascorbyl phosphate (MAP), alpha-lipoic acid (ALA) and kinetin, as well as their effects on the hydration and viscoelastic skin properties. The photoprotection was determined in vitro (antioxidant activity) and in vivo on UV-irradiated hairless mouse skin. The hydration effects were performed with the application of the formulations under study on the forearm of human volunteers and skin conditions were analyzed before and after a single application and daily applications during 4 weeks in terms of transepidermal water loss (TEWL), skin moisture and viscoelastic properties. The raw material under study possessed free-radical scavenging activity and the formulation with it protected hairless mouse skin barrier function against UV damage. After 4 weeks of application on human skin, the formulation under study enhanced stratum corneum skin moisture and also showed hydration effects in deeper layers of the skin. Thus, it can be concluded that the cosmetic formulation containing a dispersion of liposome with MAP, ALA and kinetin under study showed photoprotective effects in skin barrier function as well as pronounced hydration effects on human skin, which suggests that this dispersion has potential antiaging effects.

In vitro micro propagation of Nicotiana benthamiana via axillary shoots

Axillary shoots of Nicotiana benthamiana were regenerated from nodal explants in two weeks using MS media supplemented with the cytokinin, kinetin (0.5 mg/L), and the auxin, indole-3-butyric acid (IBA) (0.1 mg/L). Ninety two percent of shoots were 2.1-20 mm tall, a size ideal for root induction. After transfer to hormone-free MS they readily produced roots within seven days, with phenotypically normal, fully developed plants being obtained within four weeks. Leaf chlorosis due to iron deficiency was observed in plants over time, however, this was overcome by doubling the concentration of inorganic iron. This rapid micro-propagation system is particularly useful for the in vitro mass production of N. benthamiana plants for various biotechnological applications.

In vitro culture of the allergenic weed Parthenium hysterophorus L

Excised stem, leaf segments and whole flower of the allergenic weed P. hysterophorus were cultured on Murashighe and Skoog's basal medium supplemented with hormones. Shoot buds readily formed in the stem callus cultured on MS Medium supplemented with IAA and BAP or Kinetin. The leaf callus formed roots alone in a wide variety of media. Suspension cultures were initiated from the leaf and stem callus. The leaf callus elicited a positive patch test response for delayed hypersensitivity in 4 patients suffering from Parthenium dermatitis, thus indicating its ability to synthesise the allergenic principle(s).

In vitro micropropagation of elite rosewood (Dalbergia latifolia Roxb

Induction of single and multiple shoots was obtained from nodal expiants of 60–80 year-old elite trees of rosewood on Murashige and Skoog's basal medium supplemented with 6-benzylaminopurine (1.0 mg 1-1) and delta -Naphthalene acetic acid (0.05 mg 1-1) or indole acetic acid (0.5 mg 1-1). Multiplication of shoots was obtained on MS (reduced major elements) or Woody Plant Medium supplemented with 6-benzylaminopurine (1.0 mg 1-1) and kinetin (0.5–1.0 mg 1-1). Excised shoots were rooted on half-strength MS with IBA (2.0 mg 1-1) to obtain complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred to the soil.

Isolation and characterization of a cytokinin-binding protein from cucumber cotyledons

A protein which binds specifically to [3H]-zeatin has been isolated from cucumber cotyledons by chromatographic techniques. Its binding to [3H]-zeatin was inhibited remarkably by the addition of non-radioactive cytokinins and the order of inhibition was zeatin > -zeatin riboside > N6-(Delta2-isopentenyl)adenine > N6-(Delta2-isopentenyl)adenosine > N6-benzyl-adenosine > kinetin riboside. This protein behaved as a soluble protein with an apparent molecular size of 43,000 daltons on gel filtration through calibrated Sephadex G-100 column. The dissociation constant, Kd, of the protein-zeatin complex was about 4 × 10–7 M.

In vitro regeneration from immature cotyledon explant and protein profile changes during organogenesis in groundnut (Arachis hypogaea L.)

Complete plants were regenerated from in vitro cultured immature cotyledon segments of groundnut (Arachis hypogaea L. cv. TMV-7) by organogenesis. Callus cultures were best Initiated from immature cotyledon segments on MS (Murashige and Skoog) salts containing B5 vitamins supplemented with indole-3-acetic acid (IAA) and alpha -naphthalene acetic acid (NAA; 4.0 mg L-1) and kinetin (KIN; 0.5 L-1). Calluses were transferred to a medium containing KIN (2.0 mg L-1) and IAA and NAA (0.5 mg L-1) for shoot Initiation. The regenerated shoots were transferred to a medium containing Indole-3-butyric acid (IBA; 2.0 mg L-1) and KIN (0.2 mg L-1) for developing roots. In vitro produced plantlets developed sucessfully, matured, and set seed. The protein profiles [sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE)] of callus, callus with shoot, and callus with shoot and root showed differences.

The requirements for Ca2+, protein phosphorylation and concurrent protein synthesis for zeatin signaling of acidic chitinase transcript accumulation in Cucumis sativus L

We have previously reported that both Ca2+ and staurosporine-sensitive protein kinase(s) are involved in the cytokinin zeatin induction of cucumber chitinase activity and its protein content (Barwe et al. 2001). To further characterize signal transduction events involved in this cytokinin induction of chitinase gene expression, Northern hybridizations of total RNAs prepared from excised, dark-grown cucumber cotyledons treated with cytokinins and/or various agonists and antagonists of signal transduction components, were carried out using a cucumber acidic chitinase (CACHT) cDNA probe (Metraux et al. 1989). CACHT mRNA increased by approximately 5- to 6-fold in response to exogenous zeatin (Z), zeatin riboside (ZR), and benzyladenine (BA) treatment, but failed to accumulate in response to kinetin (K). Among the cytokinins tested, Z was most effective. The Z-induced accumulation of CACHT mRNA was inhibited by a plasma membrane Ca2+ channel blocker verapamil. Treatment of cotyledons with exogenous CaCl2 and calcium ionophore A23187 in the presence and absence of cytokinin enhanced CACHT mRNA accumulation. These two observations suggest the participation of extracellular calcium in signaling Z-induction. Furthermore, the presence of staurosporine (an inhibitor of protein kinase) in Z treatment reduced CACHT mRNA, suggesting the involvement of phosphorylation of one or more cellular proteins. In addition, we provide evidence that the Z-induction of CACHT mRNA is blocked by protein synthesis inhibitor cycloheximide treatment. Taken together, these results suggest that Ca2+ influx from extracellular space, protein phosphorylation, and concurrent protein synthesis events participate in cytokinin signaling during Z-induced CACHT transcript accumulation.

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.

小麦TaRanBP基因功能的初步分析

小G蛋白（small GTPases）是真核生物中广泛存在的一类调节各种生命活动的信号分子。根据结构与功能的不同，小G蛋白家族成员可分成五个亚家族，分别为Ras，Rab，Rho，Arf和Ran。五类小G蛋白通过其活化态（GTP结合态）和非活化态（GDP结合态）的相互转换行使着各种功能。Ras GTPases在酵母和哺乳动物中调节细胞增殖过程; Rho GTPases调控肌动蛋白重组过程，并参与MAP 激酶的细胞信号转导过程等; Rab GTPases和Arf GTPases分别在膜转运过程中起着不同的重要作用;而Ran GTPases则在核孔位置调节着蛋白和RNA分子的运输过程。 小G蛋白附属蛋白调节着小G蛋白活化态与非活化态之间的转换，其中鸟核苷酸交换因子(guanine nucleotide exchange factors, GEFs)可以催化小G蛋白转换为GTP结合形式，即活化态;而GTPase 激活蛋白(GTPase-activating proteins, GAPs)和小G蛋白结合蛋白(small GTPases binding proteins)可以激活小G蛋白自身的水解活性，从而将其转变成非活化态形式。 相比其它小G蛋白，Ran GTPases及其附属蛋白在真核生物中的研究相对较少。已有的成果表明它们主要在核质运输过程中及对相应的信号转导途径起调节作用。而针对Ran GTPases及其附属蛋白在真核生物尤其是高等植物个体发育过程中的作用，目前报道还很少。 为了揭示Ran结合蛋白(Ran binding protein, RanBP)在植物发育过程中的作用，本文通过转基因手段对其功能进行 了研究。在此之前，本实验室已从小麦cDNA文库中成功克隆Ran结合蛋白基因：TaRanBP。该基因cDNA全长1035 bp，编码207个氨基酸。通过农杆菌介导叶圆片法，分别用正义、反义及TaRanBP与GFP融合蛋白等表达载体转化烟草，并成功获得转基因植株。亚细胞定位观察发现TaRanBP蛋白主要定位于细胞质内，尤其是在核膜附近富集。生理学和细胞学等方面的研究分析发现，TaRanBP基因在烟草个体发育过程中产生重要作用。过量表达TaRanBP基因的转基因植株在一定数量上表现出愈合的花冠筒上出现不同程度开裂，花冠筒上有附生舌状花瓣，及带有花瓣状颜 色的花萼等异常花表型。同时，转反义基因在一定程度上促进了转基因植株初生主根的生长（为对照烟草的2.3倍），而转正义基因烟草与对照烟草的初生主根长度差异不明显。用碘化丙锭(Propidium Iodide, PI)进行根部细胞染色。观察发现，不同的转基因烟草与对照烟草之间在根的各个不同形态区域的细胞大小差异不明显，推测根长的差异可能是由于整体细胞数目变化的原因导致。向重力性实验发现，转反义基因烟草幼苗较对照烟草的向重力性反应增加，而转正义基因的则表现为降低。激素吲哚乙酸(Indoleacetic Acid, IAA)的添加处理可以恢复转反义基因烟草的向重力性异常表型，而对转正义基因烟草几乎无影响。添加激动素(Kinetin, KT)的处理发现不同转基因烟草和对照烟草的向重力性均有减弱。观测后期，转正义基因的向重力敏感性较对照烟草得到恢复。测量不同转基因株系T1代幼苗鲜重，发现不同转基因烟草和对照烟草的幼苗鲜重动态变化在各个时间点有差异，且差异情况不尽相同。而不同转基因幼苗T1代幼苗可溶性蛋白含量较对照烟草有不同程度的下降。这种下降并没有影响转基因烟草的整体生长进程，开花期和结实情况与对照烟草相比也无明显变化。

Effect of CDC25 and WEE1 on Plant Cell Cycle and Morphogenesis

The cell cycle comprise the four phases of, G1, S-phase, G2 and mitosis. Two critical transitions are G1/S and G2/M; the latter is regulated by WEE1 kinase and CDC25 phosphatases. The scope of this thesis was to investigate the regulation of the G2/M transition of the cell cycle by WEE1 and CDC25, and how these genes interface with plant growth regulators in Arabidopsis thaliana. In Arabidopsis roots, the frequency of lateral roots was found to be increased by ectopic expression of Schizosaccharomyces pombe (Sp)cdc25e and reduced by Arath;WEE1 expression. I examined the effect of Arath;WEE1 and Spcdc25 on induction of shoots and roots in Arabidopsis hypocotyls in vitro. Hypocotyl explants from two over-expressing WEE1 lines , three T-DNA insertion lines and two expressing cdc25 (Spcdc25e) lines together with wild type (WT) were cultured on two-way gradients of kinetin (Kin) and naphthyl acetic acid (NAA). Below a threshold concentration of NAA (100 ng ml-1), WEE1 repressed morphogenesis in vitro, whereas at all NAA/Kin combinations Spcdc25 promoted morphogenesis (particularly root formation) over and above that in WT. Loss of function wee1-1 cultures were very similar to WT. Quantitative data indicated a significant increase in the frequency of root formation in Spcdc25e cultures compared with WT particularly at low Kin concentrations, and WEE1oe’s repressive effect was overcome by NAA but not Kin. In conclusion, WEE1 has a repressive effect on morphogenesis in vitro that can be overcome by auxin whereas Spcd25 by-passes a cytokinin requirement for the induction of morphogenesis in vitro. The role of CDC25 and WEE1 in DNA damage responses was also analysed. Two over-expressing Arath;CDC25 lines and T-DNA mutants showed no difference to WT either in standard conditions or zeocin-supplemented treatments. However, root length was longer in Arath;CDC25oe lines treated with hydroxyurea (HU) and lateral root number was increased compared to WT. This suggests a differential response of Arath;CDC25oe in the DNA replication (HU-induced) and DNA damage (zeocin-induced) checkpoints (Chapter 5). Finally the roles of WEE1 and CDC25 in cell cycle regulation were examined using tobacco TBY-2 cell cultures expressing Arath;WEE1, Nicotiana tabacum (Nicta)WEE1 or Arath;CDC25. Whilst Nicta;WEE1 lengthened G2 of the cell cycle, Arath;WEE1 had an unusual effect of shortening G2 phase and Arath;CDC25 had no observable effect (Chapter 6).

Gene Dosage Effect of WEE1 on Growth and Morphogenesis from Arabidopsis Hypocotyl Explants.

Background and Aims How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. Methods Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1oe), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. Key Results Quantitative data indicated a repressive effect in WEE1oe and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1oe seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1oe and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1oe for all three ground tissues but for wee1-1 only cortical cell size was reduced. Conclusions There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Cyclic GMP modulates stomatal opening induced by natriuretic peptides and immunoreactive analogues

In this paper we demonstrate that compounds that promote stomatal opening such as kinetin, atrial natriuretic peptide (ANP) and plant natriuretic peptide immunoanalogues (irPNP) significantly elevate cGMP in guard cell protoplasts. Stomata opened by irPNP are induced to close in the presence of the guanylate cyclase inhibitor, LY 83583. The effect of cGMP on stomatal opening appears to be linked with Ca2+ levels. ANP, irPNP and 8-Br-cGMP all induce stomatal opening and this is inhibited by compounds that lower intracellular Ca2+ levels such as ethylene glycol bis(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), ruthenium red and procaine.