Sunflower (Helianthus annuus L.) biochemical properties and seed components affected by potassium fertilization under drought conditions.

The seed yield and healthy oil in sunflower (Helianthus annuus L.), as an important industrial crop, decrease under stress. There is not much investigation, to our knowledge, on the use of potassium fertilization, a regulator of plant water potential, affecting the biochemical properties and seed components of sunflower under drought stress. Accordingly, such parameters were investigated in a split-split plot field experiment, conducted in two different field sites (Natanz (Nt) and Eghlid (Eg), Iran), using potassium fertilization (subplots, 0, 150 and 300 kg/ha) and six drought levels (main plots) in four replicates. Although stress significantly affected sunflower biochemical properties and seed components in the two fields, the effects of stress were more pronounced in the Eg site (significant interaction of field and drought). The plant alleviated the stress by increasing the proline, oleic and linoleic acid concentrations, however, potassium fertilization also increased plant tolerance further under stress by enhancing such components compared with control. Interestingly, the Eg site was more responsive to the potassium fertilization (significant interaction of field and fertilization), as the fertilizer resulted in a higher rate of plant biochemical properties and seed components. The use of potassium fertilization at 300 kg/ha (K3) was the most effective treatment in the alleviation of stress. Interestingly, under drought stress, potassium contributed to the enhanced quantity and quality of sunflower by increasing seed components, and enhancing the biochemical properties of the plant, which can also improve crop physiological mechanisms. The results can further increase our understanding related to the effects of potassium fertilization on the yield and physiology of sunflower under drought stress. Such results are of economic, environmental and health significance.

Evaluation of in vitro genotoxic effects induced by in vitro anther culture conditions in sunflower.

Sunflower is a globally important oilseed, food, and ornamental crop. This study seeks to investigate the genotoxic effects of tissue culture parameters in sunflower calli tissues belongs to two genotypes obtained via anther culture. Anthers were pretreated with cold for 24 hours at 4°C and heat for 2 days at 35°C in the dark and plated onto media supplemented with different concentrations and combinations of 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, α-naphthalene acetic acid and indole-3-acetic acid. Obtaining calli tissues were used to detect the DNA damage levels by Comet assay, evaluating changes on superoxide dismutase and guaiacol peroxidase activities derived from in vitro culture factors. 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 2 mg/L α-naphthalene acetic acid from plant growth regulators showed acute genotoxic effect while 0.5 mg/L indole-3-acetic acid and 0.5 mg/L α-naphthalene acetic acid showed no genotoxic effect. Total protein content analysis of antioxidant enzymes revealed that although superoxide dismutase activity did not increase, Guaiacol peroxidase (GPOX) activity decreased in comparison to control. The obtained results have indicated that in vitro culture factors apparently lead to genotoxicity and oxidative stress.

Seed-specific transcription factor HSFA9 links late embryogenesis and early photomorphogenesis.

HSFA9 is a seed-specific transcription factor that in sunflower (Helianthus annuus) is involved in desiccation tolerance and longevity. Here we show that the constitutive overexpression of HSFA9 in tobacco (Nicotiana tabacum) seedlings attenuated hypocotyl growth under darkness and accelerated the initial photosynthetic development. Plants overexpressing HSFA9 increased accumulation of carotenoids, chlorophyllide, and chlorophyll, and displayed earlier unfolding of the cotyledons. HSFA9 enhanced phytochrome-dependent light responses, as shown by an intensified hypocotyl length reduction after treatments with continuous far-red or red light. This observation indicated the involvement of at least two phytochromes: PHYA and PHYB. Reduced hypocotyl length under darkness did not depend on phytochrome photo-activation; this was inferred from the lack of effect observed using far-red light pulses applied before the dark treatment. HSFA9 increased the expression of genes that activate photomorphogenesis, including PHYA, PHYB, and HY5. HSFA9 might directly upregulate PHYA and indirectly affect PHYB transcription, as suggested by transient expression assays. Converse effects on gene expression, greening, and cotyledon unfolding were observed using a dominant-negative form of HSFA9, which was overexpressed under a seed-specific promoter. This work uncovers a novel transcriptional link, through HSFA9, between seed maturation and early photomorphogenesis. In all, our data suggest that HSFA9 enhances photomorphogenesis via early transcriptional effects that start in seeds under darkness.

Molecular aspects of zygotic embryogenesis in sunflower (Helianthus annuus L.): correlation of positive histone marks with HaWUS expression and putative link HaWUS/HaL1L.

MAIN CONCLUSION: The link HaWUS/ HaL1L , the opposite transcriptional behavior, and the decrease/increase in positive histone marks bond to both genes suggest an inhibitory effect of WUS on HaL1L in sunflower zygotic embryos. In Arabidopsis, a group of transcription factors implicated in the earliest events of embryogenesis is the WUSCHEL-RELATED HOMEOBOX (WOX) protein family including WUSCHEL (WUS) and other 14 WOX protein, some of which contain a conserved WUS-box domain in addition to the homeodomain. WUS transcripts appear very early in embryogenesis, at the 16-cell embryo stage, but gradually become restricted to the center of the developing shoot apical meristem (SAM) primordium and continues to be expressed in cells of the niche/organizing center of SAM and floral meristems to maintain stem cell population. Moreover, WUS has decisive roles in the embryonic program presumably promoting the vegetative-to-embryonic transition and/or maintaining the identity of the embryonic stem cells. However, data on the direct interaction between WUS and key genes for seed development (as LEC1 and L1L) are not collected. The novelty of this report consists in the characterization of Helianthus annuus WUS (HaWUS) gene and in its analysis regarding the pattern of the methylated lysine 4 (K4) of the Histone H3 and of the acetylated histone H3 during the zygotic embryo development. Also, a parallel investigation was performed for HaL1L gene since two copies of the WUS-binding site (WUSATA), previously identified on HaL1L nucleotide sequence, were able to be bound by the HaWUS recombinant protein suggesting a not described effect of HaWUS on HaL1L transcription.

Types of oilseed and adipose tissue influence the composition and relationships of polyunsaturated fatty acid biohydrogenation products in steers fed a grass hay diet.

The current study evaluated the composition and relationships of polyunsaturated fatty acid biohydrogenation products (PUFA-BHP) from the perirenal (PRF) and subcutaneous fat (SCF) of yearling steers fed a 70 % grass hay diet with concentrates containing either sunflower-seed (SS) or flaxseed (FS). Analysis of variance indicated several groups or families of structurally related FA, and individual FA within these were affected by a number of novel oilseed by fat depot interactions (P < 0.05). Feeding diets containing SS increased the proportions of non-conjugated 18:2 BHP (i.e., atypical dienes, AD) and conjugated linoleic acids (CLA) with the first double bond from carbon 7 to 9, trans-18:1 isomers with double bonds from carbon 6 to 12, and these PUFA-BHP had greater proportions in SCF compared to PRF (P < 0.05). Enrichment of conjugated linolenic acids, AD and CLA isomers with the first double bond in position 11 or 12, and t-18:1 isomers with double bonds from carbon 13 to 16 were achieved by feeding diets containing FS, with PRF having greater proportions than SCF (P < 0.05). Principal component analysis visually confirmed interaction effects on these groups/families of FA, and further confirmed or suggested a number of relationships between PUFA-BHP. Feeding SS or FS in a grass hay diet and exploiting adipose tissue differences, therefore, present unique opportunities to differentially enrich a number of PUFA-BHP which seem to have positive health potential in humans (i.e., t11-18:1, c9,t11-18:2 and c9,t11,c15-18:3).

Toxic effect of nickel (Ni) on growth and metabolism in germinating seeds of sunflower (Helianthus annuus L.).

To assess the toxic effect of nickel (Ni) on the growth and some key metabolic processes in sunflower, varying levels of Ni as Ni(NO(3))(2) up to 60 mg L(-1) were applied once to sunflower cultivars SF-187 and Hysun-33 at sowing time in sand culture. An increase in Ni in the growth medium adversely affected growth parameters, sugar concentration (both reducing and non-reducing), as well as the activities of α-amylase and protease. It also slowed down mobilization of stored proteins and amino acids in the germinating seeds. However, an increase in the activities of α-amylase and protease was observed over time from 24 to 120 h after sowing. Cultivar Hysun-33 showed better performance than SF-187 in the presence of excess Ni. Overall, Ni-induced reduction in germination of sunflower seed appeared to be due to disturbance in biochemical metabolism as the availability of sugars for the synthesis of metabolic energy as well as necessary amino acids for the synthesis of proteins and enzymes essential for the growing embryo are generally reduced due to suppression in α-amylase and protease activities.

Repression by an auxin/indole acetic acid protein connects auxin signaling with heat shock factor-mediated seed longevity.

The plant hormone auxin regulates growth and development by modulating the stability of auxin/indole acetic acid (Aux/IAA) proteins, which in turn repress auxin response factors (ARFs) transcriptional regulators. In transient assays performed in immature sunflower embryos, we observed that the Aux/IAA protein HaIAA27 represses transcriptional activation by HaHSFA9, a heat shock transcription factor (HSF). We also found that HaIAA27 is stabilized in immature sunflower embryos, where we could show bimolecular fluorescence complementation interaction between native forms of HaIAA27 and HaHSFA9. An auxin-resistant form of HaIAA27 was overexpressed in transgenic tobacco seeds, leading to effects consistent with down-regulation of the ortholog HSFA9 gene, effects not seen with the native HaIAA27 form. Repression of HSFs by HaIAA27 is thus likely alleviated by auxin in maturing seeds. We show that HSFs such as HaHSFA9 are targets of Aux/IAA protein repression. Because HaHSFA9 controls a genetic program involved in seed longevity and embryonic desiccation tolerance, our findings would suggest a mechanism by which these processes can be auxin regulated. Aux/IAA-mediated repression involves transcription factors distinct from ARFs. This finding widens interpretation of auxin responses.

Structure of the stigma and style in sunflower (Helianthus annuus L. )

This is the first report of the ultrastructure of the stigma and style during and after anthesis in Helianthus annuus L. using light and transmission electron microscopy. The stigma is bifid with unicellular papillae. There is no secretion of lipids, carbohydrates or proteins at anthesis. The style is semisolid in the upper portion, closer to the stigma, and becomes solid below. Ultrastructural changes on cells of the stigma and the style are described. The transmitting tissue of the ovule is first evident 40 minutes after pollination and persists during the first stages of embryogenesis. Only one pollen tube per micropyle was observed growing through this tissue.(AU)

Structure of the stigma and style in sunflower (Helianthus annuus L. )

This is the first report of the ultrastructure of the stigma and style during and after anthesis in Helianthus annuus L. using light and transmission electron microscopy. The stigma is bifid with unicellular papillae. There is no secretion of lipids, carbohydrates or proteins at anthesis. The style is semisolid in the upper portion, closer to the stigma, and becomes solid below. Ultrastructural changes on cells of the stigma and the style are described. The transmitting tissue of the ovule is first evident 40 minutes after pollination and persists during the first stages of embryogenesis. Only one pollen tube per micropyle was observed growing through this tissue.

Loss of function of the HSFA9 seed longevity program.

Gain of function approaches that have been published by our laboratory determined that HSFA9 (Heat Shock Factor A9) activates a genetic program contributing to seed longevity and to desiccation tolerance in plant embryos. We now evaluate the role(s) of HSFA9 by loss of function using different modified forms of HaHSFA9 (sunflower HSFA9), which were specifically overexpressed in seeds of transgenic tobacco. We used two inactive forms (M1, M2) with deletion or mutation of the transcription activation domain of HaHSFA9, and a third form (M3) with HaHSFA9 converted to a potent active repressor by fusion of the SRDX motif. The three forms showed similar protein accumulation in transgenic seeds; however, only HaHSFA9-SRDX showed a highly significant reduction of seed longevity, as determined by controlled deterioration tests, a rapid seed ageing procedure. HaHSFA9-SRDX impaired the genetic program controlled by the tobacco HSFA9, with a drastic reduction in the accumulation of seed heat shock proteins (HSPs) including seed-specific small HSP (sHSP) belonging to cytosolic (CI, CII) classes. Despite such effects, the HaHSFA9-SRDX seeds could survive developmental desiccation during embryogenesis and their subsequent germination was not reduced. We infer that the HSFA9 genetic program contributes only partially to seed-desiccation tolerance and longevity.

Sunflower propagation.

Sunflower (Helianthus annuus L.) has been cultivated both as an oilseed and as an ornamental plant. Several protocols have been described for the micropropagation, direct plant regeneration by organogenesis being acceptable for this plant species. Besides a strong genotype dependency, the type and ontogenic stage of explants, environmental conditions of the culture, and media composition affect sunflower organogenesis. Several problems have hindered the ability to regenerate normal shoots; the most common being hyperhydricity and precocious flowering. This chapter describes a protocol for direct shoot regeneration from cotyledons developed and established in our laboratory, as well as the improvement regenerated shoot quality.

[Integumenal embryony in CMS sunflower line].

In ovules of cultural sunflower (Helianthus annuus L.) CMS line VIR 116, which were pollinated by wild perennial specie H. occidentalis, the integumentary embryos have been revealed. They are found out at 7-9 days after pollination in embryo sacs where there was no normal fertilization. The integumentary embryos arise from somatic cells of a parent organism and their occurrence can be one of the reasons matroclinal inheritances at interspecific hybrids.

Structure of the stigma and style in sunflower (Helianthus annuus L.).

This is the first report of the ultrastructure of the stigma and style during and after anthesis in Helianthus annuus L. using light and transmission electron microscopy. The stigma is bifid with unicellular papillae. There is no secretion of lipids, carbohydrates or proteins at anthesis. The style is semisolid in the upper portion, closer to the stigma, and becomes solid below. Ultrastructural changes on cells of the stigma and the style are described. The transmitting tissue of the ovule is first evident 40 minutes after pollination and persists during the first stages of embryogenesis. Only one pollen tube per micropyle was observed growing through this tissue.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus.

BACKGROUND AND AIMS: The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. METHODS: Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. KEY RESULTS: Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. CONCLUSIONS: In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

[MADS-box genes controlling inflorescence morphogenesis in sunflower].

MADS-box genes play an important role in plant ontogeny, particularly, in the regulation of floral organ induction and development. Eight full-length cDNAs of HAM (Helianthus annuus MADS) genes have been isolated from sunflower. They encode MADS-box transcription factors expressed in inflorescence tissues. In the frames of the ABCDE model, the HAM proteins were classified according to their structural homology to known MADS-box transcription factors. The HAM45 and HAM59 genes encode the homeotic C function and are involved in the control of the identity of pistil and stamens, while the HAM75 and HAM92 genes determine the A identity of floral and inflorescence meristems and petal identity. The HAM31. HAM2, HAM63, and HAM91 genes encode the B function and are involved in the formation of petals and stamens; and the HAM137 gene encodes the E function. Analysis of the expression of MADS-box genes in sunflower has demonstrated that the structural and functional differences between the ray and tubular flowers in the inflorescence could be a consequence of the lack of HAM59 expression during ray flower initiation.

Fermentation of sunflower seed hull hydrolysate to ethanol by Pichia stipitis.

Ethanol production from sunflower seed hull hydrolysate was evaluated using Pichia stipitis NRRL Y-7124. The hydrolysate prepared with 0.7 M H2SO4 at 90 degrees C was fermented as substrate in shaking bath experiments at 30 degrees C. In a group of experiments, the influence of various detoxification methods on the fermentability of hydrolysate was investigated at pH 6. Even though the ability of all employed pretreatments to enhance fermentation performance was close, the sequential application of overliming with sodium sulfite addition was the best detoxification method. Additional experiments were performed with detoxified hydrolysate to investigate the effect of shaking rate (70-130 rpm) and initial pH (5.5-7) on the fermentation. The highest ethanol level 11 gL(-1) was achieved at initial pH of 6 and 100 rpm shaking rate from a hydrolysate containing 48 gL(-1) total reducing sugar. The corresponding alcohol yield and volumetric productivity were 0.32 gg(-1) and 0.065 gL(-1)h(-1).

Temperature-dependent endogenous oxygen concentration regulates microsomal oleate desaturase in developing sunflower seeds.

Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.

Carbon conversion efficiency and central metabolic fluxes in developing sunflower (Helianthus annuus L.) embryos.

The efficiency with which developing sunflower embryos convert substrates into seed storage reserves was determined by labeling embryos with [U-(14)C6]glucose or [U-(14)C5]glutamine and measuring their conversion to CO2, oil, protein and other biomass compounds. The average carbon conversion efficiency was 50%, which contrasts with a value of over 80% previously observed in Brassica napus embryos (Goffman et al., 2005), in which light and the RuBisCO bypass pathway allow more efficient conversion of hexose to oil. Labeling levels after incubating sunflower embryos with [U-(14)C4]malate indicated that some carbon from malate enters the plastidic compartment and contributes to oil synthesis. To test this and to map the underlying pattern of metabolic fluxes, separate experiments were carried out in which embryos were labeled to isotopic steady state using [1-(13)C1]glucose, [2-(13)C1]glucose, or [U-(13)C5]glutamine. The resultant labeling in sugars, starch, fatty acids and amino acids was analyzed by NMR and GC-MS. The fluxes through intermediary metabolism were then quantified by computer-aided modeling. The resulting flux map accounted well for the labeling data, was in good agreement with the observed carbon efficiency, and was further validated by testing for agreement with gas exchange measurements. The map shows that the influx of malate into oil is low and that flux through futile cycles (wasting ATP) is low, which contrasts with the high rates previously determined for growing root tips and heterotrophic cell cultures.

Sunflower (Helianthus annuus L.).

Sunflower (Helianthus annuus L.) is considered one of the recalcitrant species in terms of transformation and regeneration. A routine transformation system of this crop requires competent cell cultures for efficient plant regeneration as well as an effective method for gene delivery. A transformation system was developed by an Agrobacterium tumefaciens-mediated method using split mature embryonic axis explants from the Ha89 genotype. Mean transformation efficiency obtained (measured as PCR+ plants/treated explants) varied from 1 to 5.2% depending on the use of the EHA105 or the C58 strain containing a plasmid with a gene of agronomic interest. The system developed has applicability to several Agrobacterium strains and plasmids with both reporter genes or genes of agronomic interest. Plants obtained with this protocol were confirmed by PCR and Southern blot. Stable inheritance of transgenes was successfully followed until generation T4 in several independent lines.