Photosynthesis,dry matter accumulation and distribution in the wild sunflower Helianthus petiolaris and the cultivated sunflower Helianthus annuus as influenced by water deficits

Summary This study reports on the effects of water deficits on photosynthesis, plant growth and carbon allocation in the wild sunflower Helianthus petiolaris and in the cultivated sunflower Helianthus annuus grown under controlled conditions in the glasshouse. Water deficits reduced the rate of net photosynthesis and the dry weight of leaves, stems, roots and reproductive parts in both species. The root-to-shoot ratio of about 0.05 in H. petiolaris was lower than the root-to-shoot ratio of about 0.15 in H. annuus. Water stress did not affect the root-to-shoot ratio, but increased the percentage of roots at depth in H. annuus. The decrease in growth induced by water deficits was a consequence of a reduction in both leaf area production and net photosynthesis. Flowering occurred earlier in H. petiolaris than in H. annuus with a consequent earlier allocation of carbon to reproductive parts in the wild compared to the cultivated sunflower. The time to budding and flowering of either species was not altered by mild water stress, but was delayed by severe water deficits. During mild water stress carbon allocation to stems decreased, but that to reproductive parts increased. When plants were severely stressed and then rewatered the proportion af carbon allocated to leaves increased and the proportion allocated to stems decreased when compared to unstressed plants. The adaptative role of these features is discussed.     

The domestication of Helianthus annuus L. (sunflower)

All modern domesticated sunflowers can be traced to a single center of domestication in the interior mid-latitudes of eastern North America. The sunflower achenes and kernels recovered from six eastern North American sites predating 3000 b.p. that document the early history of this important crop plant are reanalyzed, and two major difficulties in the interpretation of archaeological sunflower specimens are addressed. First, achenes and kernels obtained from a modern wild sunflower population included in a prior genetic study because of its minimal likelihood for crop-wild gene flow, and its close genetic relationship to domesticated sunflowers, provide a new and more tightly drawn basis of comparison for distinguishing between wild and domesticated achene and kernel specimens recovered from archaeological contexts. Second, achenes and kernels from this modern wild baseline population were carbonized, allowing a direct comparison between carbonized archaeological specimens and a carbonized modern wild reference class, thereby avoiding the need for the various problematic shrinkage correction conversion formulas that have been employed over the past half century. The need for further research on museum collections is underscored, and new research directions are identified.     

The nature of resistance to Dectes texanus (Col., Cerambycidae) in wild sunflower,Helianthus annuus

The longhorned beetle Dectes texanus LeConte is a serious pest of cultivated sunflowers, Helianthus annuus L. and soybeans, Glycine max (L.), throughout much of the American midwest. Significant yield losses arise from the pre‐harvest lodging of plants when mature larvae girdle the base of stalks in preparation for overwintering. We investigated possible reasons why infestation of wild H. annuus by D. texanus is remarkably rare, whereas 80–90% of plants may be infested in fields of cultivated varieties. When caged on wild sunflower plants in the field, laboratory‐reared, gravid females fed less and made significantly fewer ovipositional punctures than they did when subsequently caged on a cultivated plant. Furthermore, only 18.4% of ovipunctures in wild plants resulted in an egg being laid, compared with 44.9% in cultivated plants, suggesting that additional resistance factors reduce the probability of eggs being laid during ovipositor insertion. Female behaviour was not affected by adult diet prior to testing (wild vs. cultivated H. annuus petioles), nor was there any difference in response between females obtained as larvae from cultivated sunflower or from soybean. Petioles of wild H. annuus exuded more than four times the weight of resinous material when severed as did those of cultivated plants, were significantly less succulent as determined by water content, and required significantly more force to penetrate the epidermis in a standardized puncture test. We conclude that a combination of physical and chemical properties of wild H. annuus confers substantial resistance to oviposition by D. texanus and that this natural antixenosis has been inadvertently diminished in the course of breeding cultivated varieties.     

Composition of lipids from sunflower pollen (Helianthus annuus)

The contents of the pollen lipids of the sunflower Helianthus annuus are described. The major component is the seco-triterpene helianyl octanoate, followed by new beta-diketones as second major group of compounds. They exhibit a shorter chain length and often other positions of the functional group compared to already known beta-diketones. Of particular note are the 1-phenyl-beta-diketones, not previously reported from nature. Further lipid classes present are related hydroxyketones and diols. Interestingly, new beta-dioxoalkanoic acids are present in the extracts, which most likely are biogenetic precursors of the diketones. Additionally, we investigated the composition of the pollen coat which resembles the total extract, but lacks the dioxoalkanoic acids and certain estolides.     

Natural variation in gene expression between wild and weedy populations of Helianthus annuus

The molecular genetic changes underlying the transformation of wild plants into agricultural weeds are poorly understood. Here we use a sunflower cDNA microarray to detect variation in gene expression between two wild (non-weedy) Helianthus annuus populations from Utah and Kansas and four weedy H. annuus populations collected from agricultural fields in Utah, Kansas, Indiana, and California. When grown in a common growth chamber environment, populations differed substantially in their gene expression patterns, indicating extensive genetic differentiation. Overall, 165 uni-genes, representing approximately 5% of total genes on the array, showed significant differential expression in one or more weedy populations when compared to both wild populations. This subset of genes is enriched for abiotic/biotic stimulus and stress response proteins, which may underlie niche transitions from the natural sites to agricultural fields for H. annuus. However, only a small proportion of the differentially expressed genes overlapped in multiple wild vs. weedy comparisons, indicating that most of the observed expression changes are due to local adaptation or neutral processes, as opposed to parallel genotypic adaptation to agricultural fields. These results are consistent with an earlier phylogeographic study suggesting that weedy sunflowers have evolved multiple times in different regions of the United States and further indicate that the evolution of weedy sunflowers has been accompanied by substantial gene expression divergence in different weedy populations.     

Genetic characterization and molecular mapping of a chlorophyll deficiency gene in sunflower (Helianthus annuus)

A major gene controlling chlorophyll deficiency (phenotyped by yellow leaf color, yl) in sunflower was identified and mapped in an F(2) population derived from a cross between two breeding lines. Greenness degree was scored by a hand-held chlorophyll meter in the F(2) population. Leaf tissue from the parents, F(1) hybrids, and some F(2) progenies were also sampled to determine the chlorophyll content. All F(1) plants had normal green leaf color and the segregation of the plants in the F(2) population fits the monogenic ratio (chi((3:1))(2)=0.03, p>0.9), indicating that leaf color is a monogenic trait with normal green dominant over yellow leaf color in this population. The contents of chlorophyll a, chlorophyll b, and total chlorophyll in the yellow-leafed lines were reduced by 41.6%, 53.5%, and 44.3%, respectively, in comparison with those in the green-leafed lines. Genetic mapping with molecular markers positioned the gene, yl, to linkage group 10 of sunflower. An SSR marker, ORS 595, cosegregated with yl, and a TRAP marker, B26P17ga5-300, was linked to yl with a genetic distance of 4.2cM. The molecular marker tightly linked to the chlorophyll deficiency gene will provide insight into the process of chlorophyll metabolism in sunflower.     

Callus formation from isolated sunflower (Helianthus annuus) mesophyll protoplasts

Mesophyll protoplasts of the cultivated sunflower,Helianthus annuus, have been consistently found not to divide or regenerate calli, despite the efforts of several groups. In the present report, we describe the conditions for donor plant culture, protoplast isolation, and their culture that were suitable for repeated regeneration of green, nodular, vigorously growing calli from isolated sunflower mesophyll protoplasts. The best conditions for protoplast isolation employed the use of both CAYLA cellulase and CAYLA pectinase. Culture conditions were not much different from those established earlier for sunflower hypocotyl protoplasts. The most startling observation was the great variability of division frequencies between experiments even under strictly controlled, identical experimental conditions. This finding points to an important influence of a variable in the physiological state of the donor plant which is difficult to control.     

Combining-groups in cultivated sunflower populations (Helianthus annuus L.) and their relationships with the country of origin

Using four tester lines an analysis of combining abilities for seed yield, seed moisture content and seed oil content was performed on 39 cultivated sunflower populations originating from ten countries. A between-populations structure based on specific combining abilities (SCA) was designed, defining separate combining-groups for each of the four testers. This structure corresponds to the country in which the populations originated.     

Genetic control of organogenesis in cotyledons of sunflower (Helianthus annuus)

Genetic variability for regeneration ability was evaluated by studying direct organogenesis from cotyledons of thirteen genotypes including three cytoplasmic male sterile, three maintenor, three restorer inbred lines, and four F1 hybrids obtained by crosses between some of these inbred lines. The experimental design was a complete randomized block with three replications. A high genetic variability for organogenesis parameters between genotypes was observed in this study. Evidence of cytoplasmic effect and nucleo-cytoplasmic interaction for some of regeneration parameters was observed. The data also showed the importance of additive genetic control for organogenesis parameters in most genotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Inheritance and molecular mapping of a downy mildew resistance gene, Pl 13 in cultivated sunflower (Helianthus annuus L.)

The inheritance of resistance to sunflower downy mildew (SDM) derived from HA-R5 conferring resistance to nine races of the pathogen has been determined and the new source has been designated as Pl ₁₃ . The F₂ individuals and F₃ families of the cross HA-R5 (resistant) × HA 821 (susceptible) were screened against the four predominant SDM races 300, 700, 730, and 770 in separate tests which indicated dominant control by a single locus or a cluster of tightly linked genes. Bulked segregant analysis (BSA) was carried out on 116 F₂ individuals with 500 SSR primer pairs that resulted in the identification of 10 SSR markers of linkage groups 1 (9 markers) and 10 (1 marker) of the genetic map (Tang et al. in Theor Appl Genet 105:1124–1136, 2002) that distinguished the bulks. Of these, the SSR marker ORS 1008 of linkage group 10 was tightly linked (0.9 cM) to the Pl ₁₃ gene. Genotyping the F₂ population and linkage analysis with 20 polymorphic primer pairs located on linkage group 10 failed to show linkage of the markers with downy mildew resistance and the ORS 1008 marker. Nevertheless, validation of polymorphic SSR markers of linkage group 1 along with six RFLP-based STS markers of linkage group 12 of the RFLP map of Jan et al. (Theor Appl Genet 96:15–22, 1998) corresponding to linkage group 1 of the SSR map, mapped seven SSR markers (ORS 965-1, ORS 965-2, ORS 959, ORS 371, ORS 716, and ORS 605) including ORS 1008 and one STS marker (STS10D6) to linkage group 1 covering a genetic distance of 65.0 cM. The Pl ₁₃ gene, as a different source with its location on linkage group 1, was flanked by ORS 1008 on one side at a distance of 0.9 cM and ORS 965-1 on another side at a distance of 5.8 cM. These closely linked markers to the Pl ₁₃ gene provide a valuable basis for marker-assisted selection in sunflower breeding programs.     

The lipidome and proteome of oil bodies from Helianthus annuus (common sunflower)

In this paper we report the molecular profiling, lipidome and proteome, of the plant organelle known as an oil body (OB). The OB is remarkable in that it is able to perform its biological role (storage of triglycerides) whilst resisting the physical stresses caused by changes during desiccation (dehydration) and germination (rehydration). The molecular profile that confers such extraordinary physical stability on OBs was determined using a combination of ³¹P/¹H nuclear magnetic resonance (NMR), high-resolution mass spectrometry and nominal mass-tandem mass spectrometry for the lipidome, and gel-electrophoresis-chromatography-tandem mass spectrometry for the proteome. The integrity of the procedure for isolating OBs was supported by physical evidence from small-angle neutron-scattering experiments. Suppression of lipase activity was crucial in determining the lipidome. There is conclusive evidence that the latter is dominated by phosphatidylcholine (～60 %) and phosphatidylinositol (～20 %), with a variety of other head groups (～20 %). The fatty acid profile of the surface monolayer comprised palmitic, linoleic and oleic acids (2:1:0.25, ¹H NMR) with only traces of other fatty acids (C24:0, C22:0, C18:0, C18:3, C16:2; by MS). The proteome is rich in oleosins (78 %) with the remainder being made up of caleosins and steroleosins. These data are sufficiently detailed to inform an update of the understood model of this organelle and can be used to inform the use of such components in a range of molecular biological, biotechnological and food industry applications. The techniques used in this study for profiling the lipidome throw a new light on the lipid profile of plant cellular compartments.     

Vascular connections between the receptacle and empty achenes in sunflower (Helianthus annuus L.)

Empty achenes in sunflower, particularly in the centre of the capitulum, may be caused by poor vascularization. This hypothesis was tested by microscopic examination and translocation experiments. Phloem and xylem were identified by fluorescence of aniline-blue-stained callose and autofluorescence, respectively. Vascular strands that extended from the receptacle into empty achenes were regularly found in longitudinal sections. The phloem-mobile probe, carboxyfluorescein, was translocated from the receptacle to the pericarp and the testa of empty achenes. Similarly, (14)CO(2)-derived (14)C-photoassimilates moved into empty achenes. The observations suggest that empty achenes are both structurally and functionally connected with the vascular system of the receptacle. Hence, deficient vascular connections do not prevent seed filling in sunflower.     

Systemic acquired resistance in sunflower (Helianthus annuus L.)

Systemic acquired resistance (SAR) to infection by Botrytis cinerea in the leaves of sunflower (Helianthus annuus L.) plants was induced following cotyledon inoculation with B. cinerea or treatment with abiotic inducers. Salicylic acid (SA), benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA) or EDTA protected sunflower plants against Botrytis infection, that was revealed by a reduction in the number and area of the necrotic lesions in upper leaves after challenge inoculation with the pathogen. SA and BTH were more potent inducers than INA, EDTA or pre-inoculation with the fungus. In addition to resistance to B. cinerea, the upper leaves have also developed resistance to maceration by a mixture of cell wall-degrading enzymes. Calcium nitrate inhibited both the protective effect and the resistance of leaf discs to cell-wall degrading enzymes. All the tested chemicals increased the synthesis and excretion of sunflower phytoalexins--coumarins scopoletin and ayapin and induced the PR-proteins chitinase and 1,3-beta-glucanase, being the inducer effect of each activator correlated with the level of protection against B. cinerea (BTH > SA > INA > EDTA). Thus, SAR induction is mediated by general increase of plant defence responses. This is the first report on SAR in sunflower.     

Molecular mapping of three nuclear male sterility mutant genes in cultivated sunflower (Helianthus annuus L.)

The nuclear male sterility (NMS) trait is a useful tool for sunflower (Helianthus annuus L.) breeding and genetic programs. Previously, we induced NMS mutants in cultivated line HA 89. The mutants possessed single recessive genes ms ₆, ms ₇, and ms ₈, respectively, in NMS HA 89-872, NMS HA 89-552, and NMS HA 89-747. Bulked segregant analysis based on the male-fertile and male-sterile DNA pools and 560 simple sequence repeat and insertion/deletion markers randomly selected from 17 linkage groups (LGs) were used to locate ms ₆ to LG16, ms ₇ to LG6, and ms ₈ to LG5. Subsequent genotyping of three F2 populations of 88, 93, and 76 individuals confirmed their map positions. Additional polymorphic markers derived from four restriction fragment length polymorphism-converted sequence-tagged site primer pairs were identified. A partial linkage map consisting of eight markers was constructed for the ms ₆ locus, covering a region of 69.24 cM, with markers ORS807 and ORS996 flanking the ms ₆ locus at distances of 7.2 and 18.5 cM, respectively. Six markers were constructed for ms ₇, covering a region of 53.4 cM, with ORS608 and ORS1229 flanking ms ₇ at distances of 2.6 and 9.5 cM, respectively. Ten markers were constructed for ms ₈, covering a region of 18.0 cM, with six markers below ms ₈ and CRT518 above flanking ms ₈ at distances of 7.4 and 3.8 cM, respectively. The markers and mapping information will be useful for selection of the recessive NMS genes in sunflower breeding programs.     

Effect of the ferredoxin electron donor on sunflower (Helianthus annuus) desaturases

Ferredoxins are proteins that participate in photosynthesis and in other processes that require reducing equivalents, such as the reduction of nitrogen or fatty acid desaturation. Two classes of ferredoxins have been described in plants: light-regulated photosynthetic ferredoxins and heterotrophic ferredoxins whose activity is not influenced by light. Genes encoding the two forms of ferredoxin have been cloned and characterized in developing sunflower cotyledons. Here, these genes were overexpressed in Escherichia coli and they were purified by ion exchange and size exclusion chromatography to study their capacity to supply electrons to two different sunflower desaturases: soluble stearoyl-ACP desaturase from sunflower cotyledons, and membrane bound desaturase FAD7 expressed in yeast. In both cases photosynthetic ferredoxin was the form that promoted the strongest desaturase activity.     

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

Summary Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FeNaEDTA ethylenediamine tetraacetic acid ferric sodium salt - IAA indole acetic acid - MES morpholinoethane sulfonic acid - NAA 1-naphtalene acetic acid