Substrate-dependent modulation of ATPase activity by Na+ and K+ in roots of Plantago species

The equal rates of water vapour absorption by both bi- and trinucleate pollen indicate that their widely-differing rates of respiration have an intrinsic, biochemical basis. This was investigated with various metabolic inhibitors that were previously introduced into dry pollen via anhydrous acetone. The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, inhibited the O₂ uptake of rapidly respiring pollen and stimulated that of slowly respiring types to similar absolute values, that probably reflect the rates of substrate transport across the mitochondrial membranes. The extent of inhibition of the O₂ uptake by oligomycin, dicyclohexyl carbodiimide, antimycin A, and salicyl hydroxamic acid, alone and in combinations, indicates that hardly any oxidative phosphorylation and anabolic activities occur in slowly respiring, binucleate pollen species, having low-developed mitochondria and high energy charge values. The presence of the alternative pathway was insignificant. In other binucleate pollen species, characterized by recognizable mitochondria and low energy charge values, a limited ATP synthesis was established. The low energy charge values point to imbalance between phosphorylative and anabolic activities. In rapidly respiring, trinucleate pollen, containing well-developed mitochondria, a significant activity of the alternative oxidase was found. The energy charge values were high notwithstanding the large demand for ATP, mounting to 1.7 μmol h^?1 (mg pollen)^?1. In some pollen species, oligomycin highly stimulated the flow of electrons through the cytochrome pathway, which made an estimation of the ATP synthesis impossible.     

Mitochondrial development and activity of binucleate and trinucleate pollen during germination in vitro

Bi-and trinucleate pollen generally differ in the extent of their mitochondrial development at anther dehiscence and in the rate of their attainment of maximum-phosphorylative capacity during germination in vitro, as judged from experiments with representatives of both groups.The typically trinucleate pollen of Aster tripolium L. immediately respired at a high rate, maintaining a high energy charge. Mitochondria attained maximum electron-transducing capacity within 2 min of incubation, while tube growth started within 3 min. In contrast, the binucleate pollen of Typha latifolia L. only gradually reached a relatively low rate of respiration, concomitant with a temporary decrease in energy charge, upon immersion in the germination medium. Development of the mitochondrial, electrontransducing system occurred in about 75 min, after which the first pollen tubes emerged. Starting from a poor differentiation, mitochondria became increasingly normal in appearance as germination proceeded.The binucleate pollen of Nicotiana alata Link et Otto and Tradescantia paludosa Anders. et Woods. showed intermediate characteristics: Nicotiana resembled Typha but mitochondria developed at a higher rate; Tradescantia germinated more rapidly and resembled the trinucleate pollen of Aster.Inhibitors of mitochondrial or cytoplasmic protein synthesis failed to affect the development of the mitochondrial, respiratory capacities during pollen germination. It is concluded that the duration of the lag period is determined by the level and rate of mitochondrial development and not by the division of the generative cell.Abbreviations BSA bovine serum albumine - CAP D(-) threo chloramphenicol - CHI cycloheximide - DNP 2-4 dinitrophenol - EBr ethidium bromide - EC energy charge - EGTA ethyleneglycol-bis (2-aminoethyl ether) N, N-tetra-acetic acid - EM electron microscope - ETC electron transfer chain - HEPES N-2-hydroxyethyl piperazine N-2-ethane sulfonic acid - LSD least significant difference - PVP polyvinyl pyrrolidone - RCR respiratory control ratio - RH relative humidity - TCA tricarboxylic acid - TES N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid - URCI uncoupler respiratory control index (Hunter et al. 1976)     

Respiration and Vitality of Binucleate and Trinucleate Pollen

The respiration and vitality of ungerminated bi- and trinucleate pollen were studied in order to determine the influence of relative humidity and temperature on metabolic activity. The gas exchange, germination capacity and staining with tetrazolium bromide were followed under standardized conditions. A constant respiration rate occurred under conditions of high relative humidity (97%). Per mg pollen, the trinucleate grains of Compositae and Gramineae respired 2 to 3 times as intense as 6 species of binucleate grains. Per unit of pollen protein the differences were even larger. In contrast to binucleate pollen, the longevity of trinucleate pollen was very short and the ability to germinate was lost twice as fast as the respiration capacity. This limits the use of tetrazolium bromide as an indicator of viability. At reduced relative humidities respiration was strongly restricted, but the longevity of bi- and trinucleate pollen considerably increased. Pollen of Gramineae, however, was very sensitive to changes in relative humidity; short exposure to low relative humidity decreased both the vitality and the capacity to respire.     

The coexistence of binucleate and trinucleate pollen in Mitrephora macclurei Weerasooriya & R. M. K (Annonaceae)

Pollen of angiosperms may be either binucleate or trinucleate at maturity. According to Schürhoff–Brewbaker law, the binucleate state is pleisomorphic occuring in about 70% of all angiosperms, while the trinucleate state is advanced occurring in about 30% of the angiosperms. The derivation of trinucleate pollen from binucleate pollen is irreversible, thus trinucleate or mixed pollen was considered uncommon in early lineage. In the present work, using conventional paraffin section method, the coexistence of both binucleate and trinucleate pollen in the same intact anther were observed in Mitrephora macclurei, a species belonging to the eumagnoliid family Annonaceae. The increasing number of plants with trinucleate or mixed pollen discovered in early lineages, as well as climatic introduced changes in nuclear number challenge analyses of ancestral state and evolution of pollen nuclear number in angiosperms.     

The Effect of Concanavalin A on Membrane Potential and Intracellular pH during Activation In Vitro of Tobacco Pollen Grains

We studied the effects of short-term (5–10 min) treatment of Nicotiana tabacum L. pollen grains with concanavalin A (ConA) on their activation (changes in the membrane potential and intracellular pH) and germination in vitro. ConA (10–1000 g/ml) induced plasma membrane hyperpolarization in the vegetative cell and enhanced pollen grain germination. These effects depended on ConA concentration and were interrelated: the value of the membrane potential was negatively correlated with the number of pollen grains germinated for 1 h of their incubation (r = –0.96). In addition, ConA (100 g/ml) increased the intracellular pH value by 0.3 unit. All these effects of ConA are determined by its specific interaction with carbohydrate determinants because a competitive sugar methyl--mannopyranoside (0.1 M) completely blocked ConA effects. The data obtained presume that the specific receptors are present on the surface of pollen grains, evidently on their plasma membrane, and their interaction with lectins has a functional significance for pollen grain activation and germination.     

Cytology of teliospore germination and basidiospore formation inUromyces appendiculatus var.appendiculatus

Summary The cytology of teliospore germination and basidiospore formation inUromyces appendiculatus var.appendiculatus was characterized with light and fluoroscence microscopy. Meiosis of the diploid nucleus occurred in the metabasidium. The four haploid daughter nuclei migrated into the basidiospore initials where they underwent a post meiotic mitosis. Each basidiospore was delimited from the meatabasidium by a septum at the apex of the sterigma. Seventy-five percent of mature basidiospores were binucleate, 24.5% uninucleate, and 0.5% trinucleate. Mature, released basidiospores measured 16×9 m, were smooth-surfaced, and reniform to ovate-elliptical in shape.This study represents portion of a dissertation submitted by the senior author to the Faculty of Biology of the University of Constance in March, 1983, in partial fulfillment of the requirements for the degree of Doctor of Natural Sciences (Dr. rer. nat.).     

Aspects of evolutionary differentiation of theHamamelidaceae and the LowerHamamelididae

New investigations on the flower and fruit structure of extantHamamelidaceae and other LowerHamamelididae together with new finds of fossil flowers and seeds from the Upper and Lower Cretaceous provide the outline of an increasingly more differentiated picture of the early evolution of the subclass. Three patterns of valvate anther dehiscence are recognized in the subfamilyHamamelidoideae (and the subclassHamamelididae). The basic (plesiomorphic) type within theHamamelididae has 2 valves per theca. The type with 1 valve but 2 pollen sacs per theca is both consistent and exclusive for the 5 southern genera of theHamamelidaceae. They seem to be the remnants of a homogeneous group that originated before the Upper Cretaceous. This is supported by fossil hamamelidaceous flowers from the Upper Cretaceous that have thecae with 1 valve. Since several-seededHamamelidaceae predate one-seeded forms in the fossil seed record (in Europe) and the systematic structure of the one-seeded group is relatively more homogeneous, several-seeded groups are considered to be more ancient. Several parallel evolutionary trends are recognized within theHamamelidaceae as well as within the LowerHamamelididae: anther dehiscence with 2 valves per theca 1 slit or 1 valve; pollen sacs per theca 2 1; pollen tricolpate polyforate; exine coarsely reticulate finely reticulate; loss of perianth (tepals or petals and sepals) and concomitant loss of fixed number of floral organs; differentiation of exposed nectaries.     

Anther culture and androgenetic plant regeneration in buckwheat (Fagopyrum esculentum Moench)

Anther culture for haploid induction of buckwheat was studied over a period of five years. Approximately 24,000 anthers were isolated and cultured on different culture media. The regeneration capacity was generally very low. Data are presented for experiments that included 7278 anthers on which 99 calluses were formed and 20 buds regenerations were noted. Regeneration occurred most readily on gellan-gum solidified media, with 90 g l^-1 maltose, 2.5 mg l^-1 BA, 0.5 mg l^-1 IAA, and preferably in darkness. Haploid cells, as established by chromosome counts, were observed in eight regenerants. Several abnormalities of pollen development in vitro were detected. Starch presence in pollen as a possible sign of androgenic capacity was studied. Microspores in uninucleate and early binucleate stages contained only proplastids, while in adult pollen grains a number of amyloplasts were present.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - 2,4 D-2,4-dichloro-phenoxyacetic acid - IBA indole-3-butyric acid - 2iP 6- c,c-dimethylallylaminol-purine - NAA -naphthalene acetic acid     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca<Superscript>2+</Superscript> concentration

Cytosolic free calcium ([Ca²⁺]_cyt), which is essential during pollen germination and pollen tube growth, can be sensed by calmodulin-like proteins (CMLs). The Arabidopsis thaliana genome encodes over 50 CMLs, the physiological role(s) of most of which are unknown. Here we show that the gene AtCML24 acts as a regulator of pollen germination and pollen tube extension, since the pollen produced by loss-of-function mutants germinated less rapidly than that of wild-type (WT) plants, the rate of pollen tube extension was slower, and the final length of the pollen tube was shorter. The [Ca²⁺]_cyt within germinated pollen and extending pollen tubes produced by the cml24 mutant were higher than their equivalents in WT plants, and pollen tube extension was less sensitive to changes in external [K⁺] and [Ca²⁺]. The pollen and pollen tubes produced by cml24 mutants were characterized by a disorganized actin cytoskeleton and lowered sensitivity to the action of latrunculin B. The observations support an interaction between CML24 and [Ca²⁺]_cyt and an involvement of CML24 in actin organization, thereby affecting pollen germination and pollen tube elongation.     

Lily Cdc42/Rac-interactive binding motif-containing protein,a Rop target,involves calcium influx and phosphoproteins during pollen germination and tube growth

We report unique desiccation-associated ABA signaling transduction through which the Rop (Rho GTPase of plants) and its target LLP12-2 are regulated during the stage of pollen maturation and tube growth. Overexpression of LLP12-2 drastically inhibited pollen germination and tube growth. Studies on the germination inhibitors, Ca²⁺ influx blocking agents LaCl3 and EGTA and an actin-depolymerizing drug, latrunculin B (LatB), revealed that the LLP12-2-induced inhibition of germination and tube growth is significantly suppressed by LaCl₃ and EGTA in the LLP12-2-overexpressing pollen but not by LatB. These results suggested that LLP12-2 is associated with Ca²⁺ influx in the cytoplasm and may be not with actin assembly. With the addition of LaCl₃ and EGTA, LLP12-2-overexpressing pollen increased germination and tube growth compared with the one without addition, whereas pollen expressing GFP decreased germination and tube growth. Thus, an optimum level of [Ca²⁺]_cyt influx is crucial for normal germination and tube growth. Studies on the inhibitors, staurosporine and okadaic acid in the LLP12-2-overexpressing pollen, showed no appreciable increase in germination when compared with the one without addition, suggesting that staurosporine-sensitive protein kinases and dephosphorylation of phosphoproteins may be not involved in the LLP12-2 mediated germination. However, the LLP12-2-induced inhibition of tube length was slightly but significantly suppressed by staurosporine, suggesting that staurosporine-sensitive protein kinases involve in the LLP12-2-induced inhibition of tube growth.Key words: calcium influx, phosphoprotein, pollen tube growth, RIC protein, rop GTPaseRop (Rho GTPase of plants) was newly reported as a master regulator for plant signaling.^1,2 It participates in concerted actions of many signaling pathways that influence growth and development, and the adaptation of plants to various environmental situations.^3–5 In contrast to be negative regulators in ABA signaling,⁶ Rops might work as positive regulators in auxin signaling pathways.^7,8 Recently, we have reported unique desiccation-associated ABA signaling in which the LLP-Rop1 gene is not only negatively regulated by desiccation but also positively regulated by developmental cues independent of ABA during pollen maturation.⁹ Although LLP-Rop1 and its target, LLP12-2, accumulate in abundance in the matured and dried pollen upon dehydration, the activity of LLP-Rop1 and LLP12-2 is likely restricted at the stage of pollen maturation.⁹ As pollen germinates, ABA content decreases its level in the growing tube and thus, the activity of Rop is less restricted than that in the dried pollen and subsequently Rops become powerful regulators playing crucial roles during pollen tube growth.Pollen germination and tube growth are a continuous and highly polarized process characteristic of tip growth. As soon as pollen hydrates and germinates, a tip-focused cytoplasmic Ca²⁺ gradient is established and sustained while a pollen tube grows forward.^10,11 The Ca²⁺-permeable channels that modulate [Ca²⁺]_cyt influx in germinating pollen grains have been identified in Arabidopsis,^12,13 lily¹⁴ and pear.¹⁵ When a pollen tube grows, Rop-interactive Cdc42/Rac-interactive binding (CRIB) motif-containing proteins (RICs) play an important role as Rop GTPase targets and control a variety of Rop-dependent signaling pathways.¹⁶ RICs contain a CRIB motif required for their specific interaction with GTP-bound Rop. They are grouped into five classes that share little sequence similarity outside of the Rop-interactive domain.¹⁶ Different RICs expressed in various reproductive and vegetative parts of the Arabidopsis plant may act as Rop targets to control different Rop-dependent pathways in pollen tubes and in other organ development. For instances, RIC4 has been demonstrated to promote F-actin assembly, whereas RIC3 activates Ca²⁺ signaling by affecting [Ca²⁺]_cyt influx that subsequently results in F-actin disassembly in pollen tube growth.¹⁷ The two RICs, both activated by AtRop1, counteract each other to control the actin dynamics and polar pollen tube growth.¹⁷We have demonstrated that LLP12-2, a RIC protein, interacts with active LLP-Rop1 in vivo.⁹ To examine the function of LLP12-2 in the growing tubes, the purified LLP12-2 PCR product digested with XbaI and SacI was cloned into the corresponding sites of Zm13::GFP construct to generate the Zm13::GFP-LLP12-2 construct. The transient expression of GFP-LLP12-2 in pollen using particle bombardment was investigated. Pollen germination and tube length were measured after particle bombardment and subsequent in vitro germination. With the treatment of Ca²⁺ influx blocking agents LaCl₃ and EGTA, pollen expressing GFP alone significantly decreased germination and tube elongation, suggesting that a decrease in [Ca²⁺]_cyt influx may cause the inhibition of pollen germination and tube growth (Fig. 1 and 17Open in a separate windowFigure 1LLP12-2 inhibits pollen germination by regulating the calcium influx channels. Germination percentages were determined 9 h after particle bombardment and subsequent in vitro germination. Equal amounts of GFP and LLP12-2 DNA (7.5 µg) were transiently expressed in lily pollen after which pollen was treated without (control) or with either LaCl₃ (1 µM), EGTA (0.5 mM), LatB (0.05 nM), okadaic acid (5 nM) or staurosporine (1 µM) during germination.

Table 1

Effects of LaCl₃, EGTA, LatB, okadaic acid or staurosporine on the length of pollen tube expressing LLP12-2

AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING POLLEN EMBRYOGENESIS IN HYOSCYAMUS NIGER (HENBANE)

RNA synthesis during pollen embryogenesis in cultured anther segments of Hyoscyamus niger (henbane) has been followed by autoradiography of ³H-uridine incorporation. Embryogenic divisions were initiated in binucleate pollen grains in which the generative nucleus or both generative and vegetative nuclei synthesized RNA. When the first haploid mitosis in culture resulted in pollen grains with two nearly identical nuclei, those in which both nuclei synthesized RNA became embryogenic. Binucleate pollen grains in which ³H-uridine incorporation was confined exclusively to the vegetative nucleus gradually became starch-filled and nonembryogenic. Based on the degree of involvement of the vegetative nucleus in embryoid formation, some differences were noted between the counts of autoradiographic silver grains over cells cut off by the generative and vegetative nuclei during progressive embryogenesis. The possible significance of RNA synthesis in the nuclei of binucleate pollen grains in determining the pathway of embryogenic divisions is discussed.     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

SYSTEMATIC SIGNIFICANCE OF POLLEN NUCLEAR NUMBER IN EUPHORBIACEAE,TRIBE EUPHORBIEAE

Pollen nuclear number is determined in 139 species of 5 genera in the Euphorbieae, subtribe Euphorbiinae. The 111 new determinations are tabulated along with previous reports, and the results indicate that the distribution of binucleate (II) and trinucleate (III) pollen is strongly associated with the taxonomic groupings within the Euphorbieae. Although binucleate pollen is probably primitive within the tribe Euphorbieae, as suggested by the nuclear condition in Neoguillauminia, the situation in Euphorbia still requires further elucidation. Within Euphorbia, the morphologically most primitive species studied have III pollen despite the fact that II pollen is presumably the original condition for the subtribe Euphorbiinae. In Euphorbia, II pollen only is reported from nine sections and III pollen only from ten sections, while in four sections (Esula, Goniostema, Aphyllis, and Deuterocalli) both II and III pollen have been found. The New World species of Euphorbia nearly all have III pollen, whereas the vast majority of the African succulents have II pollen. The genera of New World origin, Chamaesyce and Pedilanthus, have III pollen, while the African genera Monadenium and Synadenium have II pollen. Independent derivations of III pollen from II pollen appear to have occurred in sections Goniostema, Aphyllis, and Deuterocalli (all of subg. Euphorbia). There is no evidence that reversals from III to II pollen have occurred.     

Stage-related expression of mRNAs during pollen development in lily and tobacco

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h^–1 in young binucleate cells, 138 fg · h^–1 in late binucleate cells and 56 fg · h^–1 in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.Abbreviations cDNA copy DNA - pI isolectric point To whom correspondence should be addressed.     

TheCaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants

We have evaluated the expression of the reporter -glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity—as estimated by the histochemical GUS assay—in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 mol MU mg protein^–1 h^–1 to 0.26 mol MU mg protein^–1 h^–1. Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.Abbreviations CaMV 35S Cauliflower mosaic virus promoter - GUS -Glucuronidase (EC 3.2.1.31) - MU 4-Methyl umbelliferone - nos Nopaline synthase promoter - nptII Neomycin phosphotransferase - X-Gluc 5-Bromo-4-chloro-3-indolyl--d-glucuronic acid     

Fluorescence microscopy of plastid nucleoids and a survey of nuclease C in higher plants with respect to mode of plastid inheritance

Summary Plastid nucleoids (pt nucleoids) were observed during pollen formation, or in generative cells of mature pollen grains using fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). Nuclease C activity was surveyed using SDS-PAGE and agarose gel nuclease assay methods. InMirabilis jalapa, pt nucleoids were observed both in pollen mother cells and the monocellular pollen grains after meiosis, followed by the complete disappearance both in the generative and vegetative cells at the bicellular pollen grain stage. This observation is a direct evidence of maternal plastid inheritance. By contrast, in the generative cells of mature pollen grains fromRhododendron kaempferi, Zygocactus truncatus, Oenothera laciniata, andO. speciosa, pt nucleoids were clearly observed. Thus cytological evidence convinces the mode of biparental plastid inheritance. Nuclease C activity was clearly detected both in the stamen and pistil ofM. jalapa. InR. kaempferi low nuclease C activity was detected in both organs, but the activity in the stamen was much less than in the pistil. InZ. truncatus, O. laciniata, andO. speciosa, the activities were difficult to detect in both organs. These results suggest a significant role of nuclease C for the digestion of pt nucleoids in the generative cells.Abbreviations EGTA ethylene-glycol-bis-(2-aminoethyl ether)-N, N, N, N-tetraacetic acid - DAPI 4,6-diamidino-2-phenylindole - Nuclease C Ca²⁺ dependent nuclease - SDS-PAGE SDS-polyacrylamide gel electrophoresis - pt nucleoids plastid nucleoids     

Heat stress differentially modifies ethylene biosynthesis and signaling in pea floral and fruit tissues

Key message

We identified and cloned the two precursors of miR158 and its target gene in Brassica campestris ssp. chinensis, which both had high relative expression in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility, which was caused by the degradation of pollen contents from the binucleate microspore stage. These results first suggest the role of miR158 in pollen development of Brassica campestris ssp. chinensis.

Abstract

MicroRNAs (miRNAs) play crucial roles in many important growth and development processes both in plants and animals by regulating the expression of their target genes via mRNA cleavage or translational repression. In this study, miR158, a Brassicaceae specific miRNA, was functionally characterized with regard to its role in pollen development of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Two family members of miR158 in B. campestris, namely bra-miR158a1 and bra-miR158a2, and their target gene bra027656, which encodes a pentatricopeptide repeat (PPR) containing protein, were identified. Then, qRT-PCR analysis and GUS-reporter system revealed that both bra-miR158 and its target gene had relatively high expression levels in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility and pollen germination ratio, and the degradation of pollen contents from the binucleate microspore stage was also found in those deformed pollen grains, which led to pollen shrinking and collapse in later pollen development stage. These results first shed light on the importance of miR158 in pollen development of Brassica campestris ssp. chinensis.

     

Parallel regulation of arginine transport and nitric oxide synthesis by angiotensin II in vascular smooth muscle cells role of protein kinase C

Summary Experiments were performed to characterize arginine transport in vascular smooth muscle cells (SMCs) and the effect of angiotensin II (Ang II) on this process. In addition, the role of arginine transport in the cytokineinduced nitric oxide (NO) production was assessed. Arginine transport takes place through Na⁺-independent (60%) and Na⁺-dependent pathways (40%). The Na⁺-independent arginine uptake appears to be mediated by system y⁺ because of its sensitivity to cationic amino acids such as lysine, ornithine and homoarginine. The transport system was relatively insensitive to acidification of the extracellular medium. By contrast, the Na⁺-dependent pathway is consistent with system B^0,+ since it was inhibited by both cationic and neutral amino acids (i.e., glutamine, phenylalanine, and asparagine), and did not accept Li⁺ as a Na⁺ replacement. Treatment of SMCs with 100nM Ang II significantly inhibited the Na⁺-dependent arginine transport without affecting systems y⁺, A, and L. This effect occurred in a dose-dependent manner (IC₅₀ of 8.9 ± 0.9nM) and is mediated by the AT-1 receptor subtype because it was blocked by DUP 753, a non-peptide antagonist of this receptor. The inhibition of system B^0,+ by Ang II is mediated by protein kinase C (PKC) because it was mimicked by phorbol esters (phorbol 12-myristate 13-acetate) and was inhibited by staurosporine. Ang II also inhibited the IL-1 induced nitrite accumulation by SMCs. This action was also inhibited by staurosporine and reproduced with phorbol esters, suggesting a coupling between arginine uptake and NO synthesis through a PKC-dependent mechanism. However, arginine supplementation in the medium (10mM) failed to prevent the inhibitory action of Ang II on NO synthesis. These findings suggest that although Ang II inhibits concomitantly arginine transport and NO synthesis in SMCs, the reduction of NO synthesis is not associated with alterations in the cellular transport of arginine.Abbreviations Arg arginine - Orn ornithine - HmR homoarginine - Lys lysine - Gln glutamine - Asn asparagine - His histidine - Phe phenylalanine - Leu leucine - Cys Cysteine - Ala alanine - Ser serine - Thr threonine - Glu glutamate - mAIB -methyl-aminoisobutyric acid - BCH bicycloaminoheptane     

Classical morphological features of centrospermous families

The orderCentrospermae (Caryophyllales, Chenopodiales) as treated inA. Englers Syllabus, 12th edition (1964), is compared with several other modern and older systems with the result that no less than 11–13 (and more) families are considered to be centrospermous in the strict sense; to these may be added thePolygonales and, doubtfully, thePlumbaginales andBatidales. As indicated by their name Centrospermae their main character is the central or basal placentation in combination with campylotropy (or amphitropy) of the ovules, seeds with perisperm, and coiled or curved embryos in peripheral position. Other outstanding features are found in the embryology; the ovules are bitegmic-crassinucellate, a nucellar cap is present, as well as an endostome and air spaces; the pollen is trinucleate. Anomalous secondary thickening in stems and roots often occurs. The pollen morphology, specific P-type sieve-element plastids, and the presence or absence of betalains are also important characters. Other floral features, especially the structure of the gynoecium, the androecium, the perianth and the receptacle, as well as the morphology of the inflorescences are of taxonomic importance. The putative relationships of theCaryophyllidae can perhaps best be resolved on the basis of more detailed morphological investigations (e.g. the so-called apocarpy, the development of the androecium, the pollen morphology, chromosome numbers, etc.).Presented in the Symposium Evolution of Centrospermous Families, during the XIIth International Botanical Congress, Leningrad, July 8, 1975.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.