Immunogold localization of ribulose-1,5-bisphosphate carboxylase/oxygenase in the nitrogen-fixing cyanobacterium,Plectonema boryanum

Summary Antiserum against the Calvin cycle enzyme, ribulose-1,5-bisphosphate carobxylase/oxygenase (RuBisCO), was used in conjunction with colloidal gold to localize RuBisCO in nitrogen-fixing (fix+) and nonfixing (fix–)Plectonema boryanum cells. RuBisCO antiserum consistently labeled the cytoplasm and polyhedral bodies (carboxysomes) in both fix+ and fix– cells. Through morphometry, it was determined that significantly less gold label (indicative of RuBisCO) was present in fix+ cells. This decreased RuBisCO content correlated with a decrease in net photosynthetic oxygen evolution also observed in fix+P. boryanum.Abbreviations RuBisCO Ribulose-1,5-bisphosphate carboxylase/oxygenase - fix+ nitrogen-fixing - fix– nonfixing     

Fine‐scale temporal adaptation within a salmonid population: mechanism and consequences

We demonstrate a clear example of local adaptation of seasonal timing of spawning and embryo development. The consequence is a population of pink salmon that is segmented into spawning groups that use the same limited habitat. We synthesize published observations with results of new analyses to demonstrate that genetic variation of these traits results in survival differentials related to that variation, and that density‐dependent embryo mortality and seasonally variable juvenile mortality are a mechanism of selection. Most examples of local adaptation in natural systems depend on observed correlations between environments and fitness traits, but do not fully demonstrate local adaptation: that the trait is genetically determined, exhibits different fitness in common environments or across different environments, and its variation is mechanistically connected to fitness differences. The geographic or temporal scales of local adaptation often remain obscure. Here, we show that heritable, fine‐scale differences of timing of reproductive migration in a pink salmon (Oncorhynchus gorbuscha) resulted in temporal structure that persisted several generations; the differences enable a density‐dependent population to pack more spawners into limited spawning habitat, that is, enhance its fitness. A balanced trade‐off of survivals results because embryos from early‐migrating fish have a lower freshwater survival (harsh early physical conditions and disturbance by late spawners), but emigrant fry from late‐migrating fish have lower marine survivals (timing of their vernal emergence into the estuarine environment). Such fine‐scale local adaptations increase the genetic portfolio of the populations and may provide a buffer against the impacts of climate change.     

Relationship of size at return with environmental variation,hatchery production,and productivity of wild pink salmon in Prince William Sound,Alaska: does size matter?

Pink salmon (Oncorhynchus gorbuscha) returning to Prince William Sound (PWS), Alaska, have increased to historically high levels of abundance in recent years, but average body size at return has declined. We examined how body size at return of PWS pink salmon was related to 10 biophysical factors, including the scale of hatchery production. We also examined the effect of body size at return on productivity of wild pink salmon in PWS. For the 1975–1999 brood years, we found that an index of total abundance of pink salmon in the Gulf of Alaska and sea surface temperature during the year of return best explained the variation in pink salmon body size over time. Body size at return was significantly correlated with productivity of wild pink salmon. We used stepwise-regression to fit a generalized linear version of the Ricker spawner-recruit model to determine if body size would explain significant variation in wild-stock productivity in context with other environmental variation, including hatchery production. The results indicate that variability in wild-stock productivity is primarily driven by density-independent factors in the marine environment, but that body size of wild spawners also significantly affects productivity of wild PWS pink salmon. We conclude that the success of large-scale enhancement increasing the total run in PWS may have contributed to the decline in body size because of density-dependent growth in the Gulf of Alaska. We used a simulation model to estimate the impact of hatchery-induced changes in adult body size on wild-stock production in PWS. We estimated an annual wild-stock yield loss of 1.03 million pink salmon, less than 5% of the annual hatchery return of 24.2 million adult pink salmon for brood years 1990–1999.     

Hatching time as an indicator of environmental incompatibility and outbreeding depression in intraspecific salmon hybrids

We analysed hatching times of hybrids between two spatially separated pink salmon Oncorhynchus gorbuscha populations. We repeated the experiment in independent even‐ and odd‐year broodlines. In 1996 and in 1997, we made F1 hybrids from Auke Creek (Juneau, Alaska) females and Pillar Creek (Kodiak, 1000 km away) males and F1 controls from Auke Creek parents. Families were reared and released at Auke Creek. F2 hybrids, controls, and backcrosses were made from F1 returns in 1998 and 1999. In 2001, we made F1 hybrids at Pillar Creek with native females and Auke Creek males. Pillar Creek ancestry prolonged development: At Auke Creek, hybrid families (half Pillar Creek ancestry) developed more slowly (more Accumulated Temperature Units between fertilization and hatch; P  < 0·0001) than did controls (only Auke Creek ancestry). At Pillar Creek, families with only Pillar Creek ancestry developed more slowly than did hybrids with half Auke Creek ancestry. Development times of backcrosses were intermediate between those of hybrids and controls. The variation in development times between Auke Creek and Pillar Creek pink salmon has a genetic component that probably results from local adaptation and illustrates a mechanism that can lead to outbreeding depression in intercrosses between salmon populations ( e.g. , occurring between wild and translocated stocks).     

Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins

The tomato Cf-4 and Cf-9 genes confer resistance to infection by the biotrophic leaf mold pathogen Cladosporium. Their protein products induce a hypersensitive response (HR) upon recognition of the fungus-encoded Avr4 and Avr9 peptides. Cf-4 and Cf-9 share >91% sequence identity and are distinguished by sequences in their N-terminal domains A and B, their N-terminal leucine-rich repeats (LRRs) in domain C1, and their LRR copy number (25 and 27 LRRs, respectively). Analysis of Cf-4/Cf-9 chimeras, using several different bioassays, has identified sequences in Cf-4 and Cf-9 that are required for the Avr-dependent HR in tobacco and tomato. A 10-amino acid deletion within Cf-4 domain B relative to Cf-9 was required for full Avr4-dependent induction of an HR in most chimeras analyzed. Additional sequences required for Cf-4 function are located in LRRs 11 and 12, a region that contains only eight of the 67 amino acids that distinguish it from Cf-9. One chimera, with 25 LRRs that retained LRR 11 of Cf-4, induced an attenuated Avr4-dependent HR. The substitution of Cf-9 N-terminal LRRs 1 to 9 with the corresponding sequences from Cf-4 resulted in attenuation of the Avr9-induced HR, as did substitution of amino acid A433 in LRR 15. The amino acids L457 and K511 in Cf-9 LRRs 16 and 18 are essential for induction of the Avr9-dependent HR. Therefore, important sequence determinants of Cf-9 function are located in LRRs 10 to 18. This region contains 15 of the 67 amino acids that distinguish it from Cf-4, in addition to two extra LRRs. Our results demonstrate that sequence variation within the central LRRs of domain C1 and variation in LRR copy number in Cf-4 and Cf-9 play a major role in determining recognition specificity in these proteins.     

Effects on Embryo Development Time and Survival of Intercrossing Three Geographically Separate Populations of Southeast Alaska coho salmon, Oncorhynchus kisutch

We investigated adaptive differences among three geographically separate populations of coho salmon (Oncorhynchus kisutch) by forming first generation intercrosses (hybrid lines) and comparing them to parental types (control lines). Broodstock for the experiment came from the Gastineau Hatchery in Juneau, Hidden Falls Hatchery on Baranof Island, and Neets Bay Hatchery near Ketchikan, Alaska. All were isolated hatchery populations separated by 220–400 km and derived 15–20 years previously from single local wild populations. For each population, gametes were taken from 50 mature salmon of each sex and combined to form nine lines (three control and six hybrid); each line had 50 full-sibling families which were assigned to separate cells of an incubator at Gastineau Hatchery. Embryo survival and development times were measured as indicators of locally adapted fitness traits. Two of the control lines had higher survival rates than hybrid lines formed between either of their parental populations and other populations. Differences (p < 0.05) were found between development times for control and hybrid groups, which varied by as many as 20 days between families and as many as 30 days between control lines. The intermediate expression of development time of intercrossed lines is consistent with additive genetic variation of development time between the ancestral populations of coho salmon and indicates that important genetic divergence exists between the populations. A loss of local adaptation through a change in seasonal timing of completion of embryonic development would occur in intercrosses between the populations.     

Multi-functional T-DNA/Ds tomato lines designed for gene cloning and molecular and physical dissection of the tomato genome

In order to make the tomato genome more accessible for molecular analysis and gene cloning, we have produced 405 individual tomato (Lycopersicon esculentum) lines containing a characterized copy of pJasm13, a multifunctional T-DNA/modifiedDs transposon element construct. Both the T-DNA and the Ds element in pJasm13 harbor a set of selectable marker genes to monitor excision and reintegration of Ds and additionally, target sequences for rare cutting restriction enzymes (I-PpoI, SfiI, NotI) and for site-specific recombinases (Cre, FLP, R). Blast analysis of flanking genomic sequences of 174 T-DNA inserts revealed homology to transcribed genes in 69 (40%), of which about half are known or putatively identified as genes and ESTs. The map position of 140 individual inserts was determined on the molecular genetic map of tomato. These inserts are distributed over the 12 chromosomes of tomato, allowing targeted and non-targeted transposon tagging, marking of closely linked genes of interest and induction of chromosomal rearrangements including translocations or creation of saturation-deletions or inversions within defined regions linked to the T-DNA insertion site. The different features of pJasm13 were successfully tested in tomato and Arabidopsis thaliana, thus providing a new tool for molecular/genetic dissection studies, including molecular and physical mapping, mutation analysis and cloning strategies in tomato and potentially, in other plants as well.Equal contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this workEqual contributors to this work     

Using CRISPR/Cas9 genome editing in tomato to create a gibberellin‐responsive dominant dwarf DELLA allele

The tomato PROCERA gene encodes a DELLA protein, and loss‐of‐function mutations derepress growth. We used CRISPR/Cas9 and a single guide RNAs (sgRNA) to target mutations to the PROCERA DELLA domain, and recovered several loss‐of‐function mutations and a dominant dwarf mutation that carries a deletion of one amino acid in the DELLA domain. This is the first report of a dominant dwarf PROCERA allele. This allele retains partial responsiveness to exogenously applied gibberellin. Heterozygotes show an intermediate phenotype at the seedling stage, but adult heterozygotes are as dwarfed as homozygotes.     

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium,but not nitrogen‐fixing rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad‐spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen‐associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae‐specific PRR ELONGATION FACTOR‐THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF‐Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF‐Tu‐derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR‐Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.     

Genetic linkage mapping of allozyme loci in even- and odd-year pink salmon (Oncorhynchus gorbuscha)

We constructed genetic linkage maps of allozyme loci in even- and odd-year pink salmon (Oncorhynchus gorbuscha), using the total of 320 families (each female was crossed with two different males, and 80 females and 160 males were used for each of even year and odd year). The maps include eight linkage groups involving 22 loci. We observed substantial variation in recombination frequencies among different families within broodline and between sexes within broodlines. In the linkage analysis between sAAT-3* and sMDH-B1,2*, two even-year families and one odd-year family exhibited evidence of association, but two even-year and one odd-year families did not. Recombination rate tends to be reduced in males in pink salmon. The ratio of recombination rate (female/male), which ranged from 1.7 to infinity, averaged 2.8 in the even-year crosses and 3.2 in the odd-year crosses. The linkage groups (LG) I and II involving sAAT and mAH loci, which probably duplicated in the recent tetraploidization event, and the orders of loci in the LGs I (sAAT-3* --> mAH-4*) and II (mAH-3* --> sAAT-4*) were reversed, suggesting the possible paracentric inversion during salmonid evolution after the duplication.