Biochemical analysis of four species ofCassia L. pollen

The present paper describes the comparative biochemical studies in terms of quantitative analyses of carbohydrates, lipids, proteins, free amino acids, nucleic acids, minerals, ash and moisture as well as the identification of free amino acids of pollen of four species ofCassia L. (C. alata L,C. fistula L,C. occidentalis L andC. siamea Lam.). A significant variation in the chemical constituents was observed among the four species.C. occidentalis showed the highest levels of carbohydrate (15.15%) and protein (22.45%), andC. siamea had the lowest levels of carbohydrate (7.15%), lipid (6.2%) and protein (13.85%).C. alata andC. fistula showed intermediate results. However,C. alata showed the highest amount of free amino acids (3.8%) and the least of 1.42% was found inC. fistula. Thin layer chromatography (TLC) of free amino acids of the four species showed some homology in their amino acid content, of which proline, glutamic acid, methionine and phenyl-alanine were the most dominant. The level of nucleic acids and minerals was found to be comparatively low.C. siamea andC. alata showed an exceptionally high level of ash content (8.6 and 8.8%, respectively) while moisture content varied from 8 to 11%.     

Molecular cloning,functional expression and characterization of two serine/threonine-specific protein kinases from<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> pollen

The existence of different kinds of kinases in pollen and pollen tubes suggests that kinase-mediated signaling pathways are likely involved in regulating pollen germination and pollen tube growth during the life cycle of higher plants. We have used RT-PCR and RACE to isolate full-length cDNAs for two pollen-expressed kinases, named NtPK1 and NtPK2, of Nicotiana tabacum. NtPK1 and NtPK2 encode proteins of 365 and 369 amino acids with calculated molecular masses of 39.2 kDa and 39.5 kDa, respectively, and both proteins possess the 12 sub-domains that are conserved among protein kinases. The nucleotide and deduced amino acid sequences of NtPK1 and NtPK2 share 88% and 91% identity, respectively, with the C-terminal region being the most conserved. RT-PCR analysis revealed that NtPK1 was specifically expressed in pollen and pollen tubes, and that NtPK2 was also expressed in pistil and petal. Immunoblot analysis using anti-NtPK1 and anti-NtPK2 antibodies confirmed that both NtPK1 and NtPK2 were produced in pollen and pollen tubes, and that NtPK2 was also produced in developing male gametophytes and other floral tissues. Biochemical fractionation experiments showed that, in all the tissues examined, NtPK1 and NtPK2 were present in the cytosolic fraction and not in the microsomal fraction. NtPK1 and NtPK2 were found to autophosphorylate on threonine and, for NtPK2, on serine as well. All the results taken together suggest that NtPK1 and NtPK2 are novel receptor-like cytosolic serine/threonine kinases, and could mediate signaling pathways required for pollen germination and/or pollen tube growth.The nucleotide sequence data of NtPK1 and NtPK2 reported in this paper will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession numbers AJ608156 and AJ608157, respectively     

Improvement of cotton fiber quality by transforming the<Emphasis Type="Italic"> acsA</Emphasis> and<Emphasis Type="Italic"> acsB</Emphasis> genes into<Emphasis Type="Italic"> Gossypium hirsutum</Emphasis> L. by means of vacuum infiltration

A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported. The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene -glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB. The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary. Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate. Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants. Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.Communicated by D. Bartels     

Pollen production in some plant taxa with a supposed role in allergy in Eastern India

This paper deals with the production of pollen grains per anther per flower of 15 probably allergenic pollen producing angiospermic plant taxa growing in Eastern India. The selected plant taxa includesAilanthus excelsa, Alstonia scholaris, Argemone mexicana, Azadirachta indica, Cassia alata, C. siamea, Dalbergia sissoo, Holarrhena pubescens, Lagerstroemia speciosa, Madhuca indica, Milletia pinnata, Mimusops elengi, Morus alba, M. indica andMurraya paniculata. Among the investigated taxa,A. excelsa produces the highest number of pollen grains (1635180) followed byC. siamea (1589245) andA. mexicana (463800). The production of pollen grains is lowest inH. pubescens (2500), followed byA. scholaris (5175),A. indica (6220) andM. paniculata (11200).     

Activity of salicylic acid on the growth and biochemism of Chlorella vulgaris Beijerinck

This study was conducted to investigate the influence of salicylic acid (SA) on the growth and changes of nucleic acids, protein, photosynthetic pigments, sugar content and photosynthesis levels in the green alga Chlorella vulgaris Beijerinck (Chlorophyceae). The most significant changes in the content of nucleic acids and proteins was observed at the concentration 10⁻⁴ M SA between 8 and 12 day of cultivation. This concentration of SA increased the number of cells (about 40 %) and content of proteins (about 60 %) and its secretion to the medium. The slight stimulation of protein secretion occurred on the 12th day of cultivation at concentration 10⁻⁴ M, while in the range of 10⁻⁵ M to 10⁻⁶ M the protein secretion was inhibited. SA also stimulated the content of nucleic acids, especially RNA by 20–60 %, compared with the control. The most stimulating influence upon the contents of chlorophylls a and b (50–70 %), total carotenoids (25–57 %), sugar (27–41 %) and intensity of net photosynthesis (18–33 %) was found at 10⁻⁴ M of SA. At the concentration of 10⁻⁶ M SA the slight inhibition of growth and biochemical activity of the algae was recorded at the first days of cultivation.     

Pollen dimorphism in threeTeucrium species (Lamiaceae)

In the Iberian Peninsula and Balearic Islands pollen dimorphism was found inTeucrium fruticans L.,T. pseudochamaepitys L., andT. rotundifolium Schreber. Generally, this dimorphism shows two sizes of pollen grains, the smaller more or less collapsed. The percentage of pollen viability is calculated. Differences in size between viable and nonviable pollen grains are similar, being about 40% inT. fruticans andT. pseudochamaepitys, and about 26% inT. rotundifolium. With regard to pollen viability, the percentage of male sterility is higher inT. fruticans, in which from male sterile (ms) plants, 100% nonviable pollen was obtained from every flower observed. InT. pseudochamaepitys andT. rotundifolium with rare exceptions, the percentage of nonviable pollen does not seem to be significant.     

印度血桐与中平树基因组调查及SSR分子标记分析

印度血桐与中平树是大戟科血桐属植物,该属植物具有多种药用价值,被广泛应用于民间医学中许多疾病的治疗,这两种植物种子中含有的神经酸也引起了研究者的高度关注。为确定适合印度血桐与中平树的全基因组测序研究策略,该研究采用二代高通量测序技术,结合生物信息学的方法首次测定了印度血桐与中平树的基因组大小、杂合率、重复率等基因组信息并初步分析了两种材料的SSR序列特征。结果表明:(1)印度血桐与中平树的基因组大小分别为986.84和946.23 M。(2)印度血桐与中平树的杂合率分别为0.75%和0.65%,重复序列比例分别为73.02%和71.5%。(3)通过对2种材料基因组序列的SSR特征分析,在印度血桐中共鉴定了4 499 185个SSR,在中平树中共鉴定了4 969 098个SSR。该研究结果为印度血桐与中平树SSR分子标记的筛选、开发以及全基因组深度测序提供了理论指导。     

Dioecism of the genusCallicarpa (Verbenaceae) in the Bonin (Ogasawara) Islands

The morphology and function of the flowers of three species of the genusCallicarpa, C. glabra, C. nisnimurae andC. subpubescens, endemic to the Bonin Islands, revealed that all three species were dioecious. Male plants had short-styled flowers with sterile ovaries, while female plants had long-styled flowers with non-germinating inaperturate pollen grains. Such dioecism has never been reported from the family Verbenaceae. This curious sexual system with non-germinating pollen grains may have been maintained as a reward to insect pollinators.     

芦苇腐解土中酚酸类化感物质的水分响应特性研究

芦苇和虉草均具有较强的去污能力,常作为湿地植物配置于同一人工湿地进行污水处理。芦苇作为一种强化感植物对虉草具有较强的化感作用,在自然湿地和人工湿地中均会出现芦苇代替虉草的现象,且这一现象的发生与土壤含水量存在一定联系,此外,芦苇腐解土对虉草的化感抑制效应与腐解土中总酚酸的量密切相关。为了研究芦苇腐解土中主要酚酸类物质的水分响应特性,筛选出其中对水分响应较为明显的酚酸物质种类,该研究采用高效液相色谱法,通过芦苇枯落物腐解土的制备,对不同水分环境下芦苇腐解土中酚酸类物质进行了分离和鉴定。结果表明:芦苇腐解土中可分离出没食子酸、香豆酸、香草酸、丁香酸、对香豆酸、阿魏酸、水杨酸和苯甲酸等8种酚酸类物质,其中香豆酸、苯甲酸和阿魏酸等3种酚酸类物质含量较高。分离出的8种酚酸类物质的含量与腐解土的相对含水量均呈显著线性负相关关系,即随着腐解土相对含水量的上升,酚酸类物质的含量均呈现下降趋势,且各种酚酸类物质对水分的响应趋势均可用线性方程较好地拟合。其中,香豆酸、没食子酸和阿魏酸对芦苇腐解土的水分响应最为明显。因此,可将香豆酸、没食子酸和阿魏酸作为主要调控目标,通过调控湿地土壤中水分含量,削弱芦苇对虉草的化感抑制效应,从而维持人工湿地中虉草芦苇群落的长期稳定共存。     

The pollen-specific LIM protein PLIM-1 from sunflower binds nucleic acids in vitro

The protein PLIM-1 (formerly SF3) from sunflower is expressed exclusively in mature, free pollen. It contains two LIM domains associated with an acidic C-terminus comprising six copies of the pentapeptide motif (A,T,S) (E,D) TQN. We have expressed the pollen protein as well as some of its mutant forms inEscherichia coli and have used the bacterially produced proteins to study interactions with nucleic acids. Our studies show that the protein binds DNA and RNA in vitro to form large complexes, while mutant polypeptides containing either a single LIM domain or a destabilized first or second LIM domain do not. Although these data suggest that the biological function of PLIM-1 involves interactions with nucleic acids, its role in pollen development remains unclear.     

Pollen viability and transgene expression following storage in honey

Transgenic plants of tobacco andArabidopsis that produce genetically marked pollen, expressing the reporter geneuidA (gusA), were generated to determine whether pollen proteins can be expressed and stable in honey, a potential route by which foreign proteins might enter the wider environment. Hydrated tobacco pollen was found to lose viability rapidly in honey, while pollen in the natural dehydrated form remained viable for at least several days and in some cases several weeks, as determined by FDA staining activity and germinability. Dehydrated pollen was found to be capable of transient foreign gene expression, following microprojectile bombardment, after incubation in honey for at least 120 h. PCR amplification of transgene sequences in pollen of transgenic plants revealed that pollen DNA can remain relatively intact after 7 weeks in honey. GUS enzyme activity analysis and SDS-PAGE of pollen proteins revealed that foreign and native pollen proteins are stable in pollen incubated in honey for at least 6 weeks. We conclude that pollen may represent an ecologically important vector for transgenic protein products.     

Natural enemies of the pumpkin caterpillarDiaphania indica [Lepidoptera: Pyralidae] in Tamil Nadu

The survey for the natural enemies associated with the pumpkin caterpillar,Diaphania indica revealed the presence of 20 species of parasitoids, predators and pathogens. Of these, 16 were parasitoids belonging to the familiesBraconidae, Ichneumonidae, Bethylidae, Elasmidae andChalcididae. Except for 3 species the remaining parasitoids were new records forD. indica. The predators recorded were ants and spiders. A microsporidia also was recorded for the first time onD. indica.      

A synopsis of the genus allomarkgrafia (Apocynaceae)

The nine species of the genusAllomarkgrafia are treated synoptically, with a review of their systematics and nomenclature. Two new species,Allomarkgrafia ecuatoriana andA. insignis, and a new combination,Allomarkgrafia campanulata, are proposed here.     

Distyly and pollen dimorphism inLinum suffruticosum (Linaceae)

The Mediterranean distylousLinum suffruticosum has dimorphic pollen. That from short-styled plants has sub-monomorphic surface excrescences with minute papillae; that from long-styled plants has strongly dimorphic excrescences with conspicuous papillae. The differences are much like those described in species of other sections of the genus. Pollen of the related homostylousL. tenuifolium has monomorphic pollen. It combines characteristics of bothL. suffruticosum types but resembles that of the long-styled plants somewhat more closely. Ribbonlike styles of long-styled plants ofL. suffruticosum provide another feature which seems to be controlled by the S supergene thought to be associated with distylic incompatibility. The pollen ofL. suffruticosum andL. tenuifolium is very similar to that of some species of sectionLinastrum and would support their placement in that section.     

Essential amino acids of starch synthase IIa differentiate amylopectin structure and starch quality between <Emphasis Type="Italic">japonica</Emphasis> and <Emphasis Type="Italic">indica</Emphasis> rice varieties

Four amino acids were variable between the ‘active’ indica-type and ‘inactive’ japonica-type soluble starch synthase IIa (SSIIa) of rice plants; Glu-88 and Gly-604 in SSIIa of indica-cultivars IR36 and Kasalath were replaced by Asp-88 and Ser-604, respectively, in both japonica cultivars Nipponbare and Kinmaze SSIIa, whereas Val-737 and Leu-781 in indica SSIIa were replaced by Met-737 in cv. Nipponbare and Phe-781 in cv. Kinmaze SSIIa, respectively. The SSIIa gene fragments shuffling experiments revealed that Val-737 and Leu-781 are essential not only for the optimal SSIIa activity, but also for the capacity to synthesize indica-type amylopectin. Surprisingly, however, a combination of Phe-781 and Gly-604 could restore about 44% of the SSIIa activity provided that Val-737 was conserved. The introduction of the ‘active’ indica-type SSIIa gene enabled the japonica-type cv. Kinmaze to synthesize indica-type amylopectin. The starch in the transformed japonica rice plants exhibited gelatinization-resistant properties that are characteristic of indica-rice starch. Transformed lines expressing different levels of the IR36 SSIIa protein produced a variety of starches with amylopectin chain-length distribution patterns that correlated well with their onset temperatures of gelatinization. The present study confirmed that the SSIIa activity determines the type of amylopectin structure of rice starch to be either the typical indica-type or japonica-type, by playing a specific role in the synthesis of the long B₁ chains by elongating short A and B₁ chains, notwithstanding the presence of functional two additional SSII genes, a single SSI gene, two SSIII genes, and two SSIV genes in rice plants.     

Transgenic ryegrasses (Lolium spp.) with down-regulation of main pollen allergens

Ryegrass pollen (Lolium species) is a widespread source of air-borne allergens and is a major cause of hayfever and seasonal allergic asthma, which affect approximately 25% of the population in cool temperate climates. The main allergens of ryegrass pollen are the proteins Lol p 1 and Lol p 2. These proteins belong to two major classes of grass pollen allergens to which over 90% of pollen-allergic patients are sensitive. The functional role in planta of these pollen allergen proteins remains largely unknown. Here we describe the generation and analysis of transgenic plants with reduced levels of the main ryegrass pollen allergens, Lol p 1 and Lol p 2 in the most important worldwide cultivated ryegrass species, L. perenne and L. multiflorum. These transgenic plants will allow the study of the functional role in planta of these pollen proteins and the determination of potential for development of hypo-allergenic ryegrass cultivars.     

Cytotaxonomic study on two putative hybrids in the genusDuchesnea (Rosaceae)

The karyotypes and chromosome associations at meiosis in two types of natural hybrids, 7x and 8x, betweenDuchesnea chrysantha (2x) andD. indica (12x) were investigated. The 7x hybrid had a haploid chromosome set from each parent plant, whereas the 8x hybrid was considered to have a full set ofD. chrysantha and half a set ofD. indica. In the two hybrids, the chromosomes ofD. chrysantha andD. indica conjugated only slightly at meiosis. It is probable that no common genome set between the diploidD. chrysantha and the dodecaploidD. indica exists. The present evidence indicates thatD. chrysantha andD. indica should be considered to be distinct species, although they have sometimes been treated as a single species.     

Immunocytochemical distribution of polygalacturonase and pectins in styles of distylous and homostylous Turneraceae

Distyly is a plant breeding system in which two self-incompatible, but cross-compatible, floral morphs occur within populations. The morphs differ in having a reciprocal arrangement of styles and anthers. Little or nothing is known of the proteins involved in self-incompatibility for any distylous species. Here we show that a 35 kDa putative polygalacturonase is specific to the transmitting tissue of short-styled plants of five species in series Turnera. The polygalacturonase was not detected in styles of long-styled plants, or in styles of five homostylous self-compatible species in this series of the genus. It is also absent from two X-ray generated mutants and a spontaneous somatic homostylous mutant that arose on a short-styled plant and whose style does possess this polygalacturonase. Three more distantly related species in the Turneraceae were investigated. Turnera weddelliana (series Salicifoliae) does possess the polygalacturonase; however, T. diffusa (series Microphyllae), and Piriqueta caroliniana, showed no evidence of possessing this polygalacturonase using immunocytochemistry. Polygalacturonase assays revealed activity in styles of long- and short-styled plants, but showed no activity of the 35 kDa style polygalacturonase. The distribution of pectins in styles and pollen tubes revealed no difference between the long- and short-styled morphs. Methyl-esterified pectins occur throughout the style tissues, except in the transmitting tissue. The transmitting tissue possesses unesterified pectins that could provide a substrate for polygalacturonase activity. We propose that the style polygalacturonase might act in a complementary manner, allowing pollen of long-styled plants to grow through short styles or, alternatively, oligogalacturonide products of polygalacturonase activity might play a role in signalling compatible responses.     

A preliminary survey of airborne pollen in Madras City

The atmospheric pollen of Madras city was surveyed during a 1-year period (January–December 1995) with a vertical cylinder trap. A total of 32 pollen types were identified, among which nine were present throughout the year. These belonged to Poaceae,Casuarina equisetifolia Foster and Foster f.,Prosopis juliflora (SW.) DC.,Acalypha indica L.,Parthenium hysterophorus L., Cyperaceae,Cocos nucifera L., Amaranthaceae, andTypha angustata Borry and Chaub. Among the identified pollen, 61.05% belonged to trees, 21.01% to grasses, 11.65% to herbs and 6.27% to shrubs. Anemophilous pollen contributed about 52.87% to the total while entomophilous and amphiphilous pollen contributed 38.89 and 8.22%, respectively. Pollen belonging to Poaceae were found to be most predominant in the air of Madras city followed byCasuarina equisetifolia andProsopis juliflora.     

Influence of arbuscular mycorrhizae on the metabolism of maize under drought stress

A greenhouse experiment was carried out to investigate the influence of the arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) on metabolic changes in tropical maize (Zea mays L.) under drought. Two cultivars, Tuxpeno sequia CO (drought sensitive) and C8 (drought resistant), were subjected for 3 weeks to water stress following tasselling (75–95 days after sowing). Fully expanded 7th or 8th leaves were sampled and assessed for levels of chlorophyll, sugars, proteins, and amino acids. Chlorophyll content was not altered either by water stress or the presence of mycorrhizae. Mycorrhizal plants (M+) had higher total and reducing sugars than nonmycorrhizal plants (M-) at the end of 3 weeks of the drought cycle. An increase in protein content was observed with drought stress in M + plants of the cultivar C0. Most of the amino acids showed a linear increase during the period of water stress in M+ and M- plants for both cultivars. Total amino acids increased by 40.6% and 43.7% in M- plants of C0 and C8, respectively. With the presence of AM fungus, amino acid levels increased by only 10.7% and 19.2% of leaf dry mass in C0 and C8, respectively. Alanine, asparagine, glutamine, and glycine accounted for 70% of the amino acid pool. Under drought, AM inoculation enabled the plants to retain considerable amounts of sugars and proteins, especially in the drought-sensitive cultivar C0. This may be of physiological importance in helping the plant to withstand moderate drought.