Protoplasts from cotyledon and hypocotyl of sunflower (Helianthus annuus L.): shoot regeneration and seed production

Summary Cotyledon and hypocotyl protoplasts of Helianthus annuus inbred line 47 302 bcd were embedded in alginate and plated on L4 medium (Lenée and Chupeau 1986). After one month, the calli were transferred on MSSH regeneration medium (Murashige and Skoog 1962; Schenk and Hildebrandt 1972) where they regenerated shoots (overall efficiency 10^–2%). The shoots were elongated on B5 (Gamborg et al. 1968) medium first without hormones, then supplemented with GA3 and BAP (both 0.05 mg/l). In order to overcome the difficulty to induce rooting by classical methods, the elongated shoots were grafted on a sunflower rootstock. The grafted shoots produced flowers and seeds. Different factors have been shown to have an important influence on the capacity to regenerate shoots: the genotype, the physical culture conditions at the callus regeneration step (e.g. protoplasts embedded in alginate), and the media composition.Abbreviations BAP 6-benzylaminopurine - GA3 gibberellic acid - IBA indole-3-butanoic acid - IAA indole acetic acid - MES 2-N-morpholinoethane sulfonic acid - NAA 1-naphthalene acetic acid - 2,4D 2,4 dichlorophenoxyacetic acid     

Autonomous changes in the orientation of cortical microtubules underlying the helicoidal cell wall of the sunflower hypocotyl epidermis: spatial variation translated into temporal changes

Summary. Angles (λ) at which parallel cortical microtubules (cMTs) were oriented with respect to the longitudinal direction were measured in Helianthus annuus hypocotyl epidermal cells. Histograms showing λ frequencies in cell populations at the instant of epidermis fixation were obtained. Analysis of the histograms indicates that, in a particular position within a cell, the angle λ changes periodically with time, i.e., there is a cycle of λ change at that position. This cycle is most likely rotational rather than oscillatory, i.e., the change in λ has a defined chirality (clockwise or counterclockwise). The full diversity of histograms can be consistently explained by rotational cycles with a variable velocity of λ change, and with a cMT rebuilding stage taking place at a different phase of the cycle. The rotational cycles also provide the simplest explanation of cMT arrays in which the angle λ changes along a cell (fixed) and no parallel orientation of cMTs is apparent at a certain position. This explanation assumes a gradient in the phase of the rotational cycle along the cell. The symmetry of the angular characteristics of the rotational cycle, with respect to the morphological directions in cells, leads to the concept that these directions typically represent the principal directions of a certain tensor quantity, which may control the cycling. Possible interactions between the rotational cycle of cMT reorientation and the helicoidal cycle during cell wall formation are discussed. Correspondence and reprints: Department of Biophysics and Cell Biology, Silesian University, ulica Jagiellonska 28, 40-032 Katowice, Poland.     

Exposure to low levels of photosynthetically active radiation induces rapid increases in palisade cell chloroplast volume and thylakoid surface area in sunflower (Helianthus annuus L.)

Summary Sudden changes in photoactive radiation (PAR) (wavelength, 400–700 nm) induces rapid surface area changes in chloroplast thylakoid membranes. Although this response may have important photo-acclimative functions for the plant, little is known about the mechanisms by which changes in irradiance are detected or how thylakoid membranes actually increase or decrease surface area. Knowledge of the time required for significant changes in thylakoid area would help eliminate or support several possible mechanisms that may be involved in this aspect of photo-acclimation in plants. Leaf tissues were acclimated to a PAR of 500 mol quanta per m² per s then exposed to low irradiance (PAR, 50 mol quanta per m² per s) and sampled at 5, 15, 30, and 60 min post exposure. Tissue and cell structure were quantified and results showed a significant increase in the surface-to-volume ratio and surface area per unit of standard leaf volume for both appressed and nonappressed thylakoids within 5 min of exposure to low irradiance. On the basis of the ratios of appressed to nonappressed thylakoids, the surface area of the nonappressed thylakoids was found to increase faster than that of the appressed thylakoids throughout the sample period. The portion of the appressed thylakoids in contact with the stroma was defined as margin thylakoids. Margin thylakoid surface-to-volume ratio did not change relative to the high-irradiance control during the sample period but did remain significantly lower than the low-irradiance control during the sample period. The ratio of appressed to margin thylakoids indicated a broadening and shortening of the appressed thylakoid stack within the first 5 min of low-irradiance exposure. The rapidity of the shade response indicates that the early events in this response probably do not directly involve gene activation pathways.Abbreviations PAR photosynthetically active radiation - Sv surface to volume density - Vv volume density - UV-B ultraviolet B radiation     

Intra-individual heterogeneity of rDNA allows the distinction between two closely related species in the Genus Helianthus

The Ribosomal DNAs of Helianthus annuus and H. argophyllus were analysed. Total DNA from single individuals of six cultivated lines, one wild ecotype ofH. annuus, and three ecotypes of H. argophyllus, were digested with various restriction enzymes. Hybridisation of Southern blots with sunflower ribosomal probes containing most of the interspacer regions (R3) or the 25 s coding region (R2) reveals different patterns from those expected: while no difference between H. annuus and H. argophyllus had been observed in previous rDNA RFLP analysis, our study clearly distinguished the two species on the basis of two different patterns when using R3 and BamHI, BstYI, or EcoRI/BamHI. Furthermore, the sum of the fragment weights of the BamHI restriction patterns was much greater than that of the rDNA entire unit-weight space. The co-existence of different rDNA units within single individuals is proposed as a model to explain these results. Four rDNA units were distinguished, which differed in their state of methylation and by the presence of mutations at two BamRI restriction sites. H. annuus individuals displayed two types of rDNA units while H. argophyllus individuals displayed four types.     

中国2种归化植物新记录

报道了发现于辽宁省的2种中国归化植物新记录:白毛马鞭草(Verbena stricta Vent.)和向日葵(Helianthus annuus L.),白毛马鞭草穗状花序粗壮而紧凑,单生枝顶或呈简单的聚伞状或圆锥状排列,且花序轴在果期显著延长,叶卵圆形,无柄,与中国有报道的本属其他物种区别明显。该种原产美国西部至中部地区,近来在辽宁省铁岭市发现有归化。向日葵原产于北美地区,被发现广泛归化于辽宁省大连市,营口市鲅鱼圈也有发现。归化种群形态特征与该地区栽培类型完全不同,其花序极多分枝,头状花序直径较小,瘦果极小,表面纹饰多样性很高。     

Plant growth in relation to ontogenetic changes in productive characteristics of individual leaves I. growth and productive characteristics ofHelianthus annuus andZinnia elegans leaves

Growth and productive characteristics of successive leaves were ontogenetically examined using sunflower and zinnia plants grown in the field. For the purpose of more advanced comprehension, ontogenetic behavior of productive characteristics was formulated with the schematic leaf growth model. The largest leaf in zinnia appeared around the middle leaf order, but did not show the highest photosythetic rate. The decreasing pattern of relative leaf area expansion rate did not significantly differ among individual leaves, and seemed inherent to species. The ontogenetic changes in net photosynthetic rate per unit leaf area showed convex curves, while those in dark respiratory rate per unit leaf area rapidly decreased with leaf development and then became constant. In part, the rapid increase of photosynthetic rate in young leaves was supported by enhancement of light utilization efficiency, along with increase of chlorophyll content. The approach of leaf angle to the horizontal was more or less accompanied by photosynthetic development and leaf expansion. It was suggested that photosynthetic maturation in leaves of the sun-leaf type appears at leaf age equivalent to 0.35 to 0.46 of leaf life span. Ontogenetic pattern of all productive characteristics basically differed little among successive leaves.     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Relationships between root permeability to water,leaf conductance and transpiration rate in sunflower (Helianthus annuus L.) cultivars

Summary The relations between leaf conductance (g_l) transpiration rate and root permeability to water (R_p) of three sunflower (Helianthus annuus L.) cultivars grown in a controlled environment cabinet are described.No differences in transpiration rates were found but it was shown that plants with low values of R_p have active stomatal closure with favourable consequences for water use efficiency under water limiting conditions.R_p was estimated by applying hydrostatic pressure on the root system. Values of R_p per unit root volume ranged from 0.34×10^–5 to 16.75×10^–5 (s MPa^–1). There were significant inter-cultivar differences (P<0.05) in R_p and g_l and an inverse correlation between R_p and the maximum values cf g_l within cultivars.Pressure applied on the root system is proposed as a useful tool for the determination of differences in the root permeability to water amongst sunflower cultivars.     

Plant donor tissue and isolation procedure effect on early formation of embryoids from protoplasts of Helianthus annuus L.

The effects of factors influencing sunflower protoplast isolation yield, plating efficiency (PE) and the early differentiation into embryoids (embryogenic capacity, EC) have been studied. Only hypocotyl-derived protoplasts divided. The variations of PE and EC in the various treatments did not seem to be linked to the protoplast yields. From statistical analysis of the data, we concluded that, the sunflower genotype, the age and height of seedlings, the part of hypocotyl used, the incubation time (from 6 to 16 hours) in enzymes of explants or of protoplasts alone, influenced PE but large variations were detected for EC. A comparison of the factors effecting EC suggested an origin, inside the hypocotyl, of cells able to give rise, after induction, to embryogenic protoplasts.     

The control of flower initiation by gibberellin in Helianthus annuus L. (sunflower), a non-photoperiodic plant

The involvement of gibberellins in the control of flowering of sunflower was studied by direct application of GA₃ to the apex of the plants, analysis of the endogenous levels of gibberellin-like substances at different plant ages, and indirectly by the application of paclobutrazol, an inhibitor of gibberellin synthesis. GA₃ speeded-up flower initiation and floral apex development. The time of GA₃ application was more critical than the amount of GA₃ applied. The endogenous levels of gibberellin-like compounds increased significantly by day 15 after sowing. The application of paclobutrazol markedly delayed floral initiation and this effect was also depedent on plant age. Both GA₃ and paclobutrazol had their greatest effects between 10 and 20 days after sowing suggesting that an increase in gibberellins in that time period plays a role in floral initiation.     

A novel feature of structural variegation in leaves of the tropical plant Schismatoglottis calyptrata

We report a novel feature of leaf variegation. As is often the case in tropical forest floor herbs, Schismatoglottis calyptrata leaves feature structural variegation. Examination of leaf anatomy in S. calyptrata revealed a novel feature of structural variegation, which was generated by variation in the spatial arrangement of the adaxial-most tip of the palisade cells. In fully green leaf parts, contact between the adaxial-most tip of the palisade cells and the cone-shaped base of the outer epidermis cells was tight, and palisade cells were arranged radially around each epidermal cell. In dull, grayish-green leaf parts, the contact was loose, and no particular spatial arrangement of palisade cells relative to epidermal cells was observed. This feature of structural variation could be disadvantageous for photosynthesis efficiency in view of the hypothesis that, for rainforest herbs, cone-shaped epidermal cells may function as lenses. However, the high frequency of leaf variegation of S. calyptrata in natural habits suggests that this structural variegation plays an unknown advantageous role.     

Effects of low and high levels of magnesium on the response of sunflower plants grown with ammonium and nitrate

The effect of the nitrogen source (ammonium and nitrate) and its interaction with magnesium on various physiological processes was studied in sunflower plants (Helianthus annuusL.). Plants were grown in hydroponic culture with nitrate (5 mM) or ammonium (5 mM) and four concentrations of magnesium (0.1, 0.8, 5 and 10 mM). After 2 weeks, growth, gas exchange and fluorescence parameters, soluble carbohydrates, free amino acids, soluble protein and mineral elements were determined. Ammonium nutrition resulted in a reduction of dry matter accumulation, as well as in a decrease in the CO₂ assimilation. Moreover, ammonium-fed plants showed a greater content of free amino acids, soluble protein, Rubisco and anions, and a lower cation content, mostly Mg²⁺. The presence of high levels of Mg²⁺ in the nutrient solution containing NH₄ ⁺ resulted in a stimulation of growth and CO₂ assimilation to the levels observed in nitrate-fed plants. The lower photosynthetic rate of ammonium-fed plants grown with low level of magnesium does not seem to be due to a lower photosynthetic pigment content, or a deficiency in Photosystem II activity, or to lower Rubisco content. Hence, Rubisco activity or other enzymes involved in CO₂ fixation could have been affected in ammonium-fed plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

向日葵AGO和DCL基因家族的鉴定和表达分析

为了解向日葵(Helianthus annuus)的Argonaute (AGO)和Dicer-like (DCL)的功能,利用拟南芥(Arabidopsis thaliana)的AGO和DCL基因序列在向日葵基因组数据库中进行同源比对,对向日葵AGO和DCL家族成员进行生物信息学分析。结果表明,从向日葵中鉴定到15个Ha AGOs和5个HaDCLs家族成员;这2类基因在染色体上的分布均不均匀。系统发育分析表明,与拟南芥相似,HaAGO家族成员可分为3个分支,HaDCL可分为4个分支;所有的HaAGO都具有保守的N domain、DUF1785、PAZ和PIWI结构域,Ha DCL家族成员都含有PAZ和RIBOc结构域。表达分析表明,Ha DCL3a和Ha DCL3b在茎和花序中高度表达;亚细胞定位表明Ha AGO多定位于细胞核。这表明向日葵中可能存在典型的RNAi干扰机制,并可能参与了协调向日葵的生长发育过程。     

Dissociation-recombination of intergenic sunflower alcohol dehydrogenase isozymes and relative isozyme activities

Two unlinked genes, Adh₁ and Adh₂, control the production of alcohol dehydrogenase (ADH) in seeds of the annual sunflower (Helianthus annuus). Each gene is polymorphic, having F and S alleles. Starch gel electrophoretic zymograms of the four possible double homozygotes have three bands, representing two homodimers and an intermediately migrating intergenic isozyme. Zymograms of double heterozygotes consist of nine bands produced by ten isozymes: six intragenics and four intergenics, two of which are coincident. Results of dissociation-recombination (D-R) experiments are reported which demonstrate the subunit composition of the intergenic isozymes, thus supporting the relationships suggested by genetic studies. Densitometric tracings of the zymogram of a cleared gel and measurements of activities of homodimer isozymes eluted from gels following D-R of an intergenic isozyme showed that the Adh₂ isozymes were more than twice as active as those of Adh₁. Measurements of activities of crude extracts from the four possible double homozygous genotypes indicated that the seeds of the genotype Adh₁^F/Adh₁^F, Adh₂^S/Adh₂^S produced more activity than the other three. This genotype is the most common one found in wild and cultivated stocks. Isozymes eluted following electrophoresis of the same extracts had averages of 19%, 70%, and 11% of total activity contributed by the Adh₁, Adh₂, and intergenic isozymes, respectively. A simple but efficient method of isozyme elution from starch gels is described which resulted in nearly full expected recovery (approximately 46%) of the ADH activity in the applied sample.Supported by Graduate School and BioMed grants and by NSF Grant GB35853.     

Light-associated nitrogen distribution profile in flowering canopies of sunflower (Helianthus annuus L.) altered during grain growth

In vegetative canopies of many species, the vertical gradient of lamina nitrogen concentration (NW) parallels the profile of light distribution in such a way that the actual nitrogen partitioning approaches the optimum pattern for canopy photosynthesis. This paper evaluates the hypothesis that a strong sink for nitrogen, viz. growing grain, affects the pattern of lamina nitrogen distribution usually described for vegetative canopies. The light and NW profiles of sunflower (Helianthus annuus L.) crops were characterised from anthesis to physiological maturity. The factorial combination of two plant populations (2.4 and 4.8 plants m^–2) and two levels of nitrogen supply (0 and 5 g N m^–2) were the sources of variation for NW and light profiles. Before the onset of nitrogen accumulation in grain, the pattern of NW was similar to that described for other species and it was related to the distribution of light in the canopy. Important changes in the profile of NW occurred during grain filling that were unrelated to the light regime. Nitrogen was mobilised from leaves in all positions in the canopy and the rate of NW change was greater in leaves closer to the grain, which were also the leaves where nitrogen was more concentrated. It is concluded that the physiological mechanisms involved in determining the distribution of leaf nitrogen in vegetative canopies do not apply to sunflower during grain filling.     

Cytological changes in palisade cells of developing sunflower leaves

Summary Changes in the relative volume of palisade cells (V_v) allocated to various organelle compartments during postemergent leaf development was measured using stereological techniques. The surface to volume ratios (S_v) of the chloroplast and mitochondrial membranes were also measured at each stage. Three leaf stages were sampled, each was defined based on lamina length (10, 45, and 150 mm). The last stage represented a fully expanded leaf. Chloroplast and nuclear compartment V_v values changed significantly in the early stages when cells were actively dividing. Mitochondrial and vacuolar compartment V_v values showed significant changes in the latter stages during cell expansion. The oil vesicle and microbody compartments showed no significant change in V_v value during the developmental process. The surface to volume ratios of the chloroplast membranes increased significantly throughout all stages of the leaf development while mitochondrial cristae S_v values did not change. Organelle replication rates appeared to be independent of changes in cell volume with each organelle exhibiting a specific replication activity pattern. The results of this study suggest two possible mechanisms for the control of cell structural development involving both intrinsic and extrinsic factors.