MMK2, a novel alfalfa MAP kinase, specifically complements the yeast MPK1 function

Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases that are activated in response to a variety of stimuli. Here we report the isolation of an alfalfa cDNA encoding a functional MAP kinase, termedMMK2. The predicted amino acid sequence ofMMK2 shares 65% identity with a previously identified alfalfa MAP kinase, termedMMK1. Both alfalfa cDNA clones encode functional kinases when expressed in bacteria, undergoing autophosphorylation and activation to phosphorylate myelin basic protein in vitro. However, only MMK2 was able to phosphorylate a 39 kDa protein from the detergent-resistant cytoskeleton of carrot cells. The distinctiveness ofMMK2 was further shown by complementation analysis of three different MAP kinase-dependent yeast pathways; this revealed a highly specific replacement of the yeastMPK1 (SLT2) kinase byMMK2, which was found to be dependent on activation by the upstream regulators of the pathway. These results establish the existence of MAP kinases with different characteristics in higher plants, suggesting the possibility that they could mediate different cellular responses.     

Megagametophyte organization in diploid alfalfa meiotic mutants producing 4n pollen and 2n eggs

Megagametogenesis was studied in five diploid alfalfa mutants producing 4n pollen and 2n eggs, using a stain-clearing technique. All mutants produced embryo sacs with a variable number of supernumerary nuclei both at the early (bi- and tetra-nucleate) and at the late (eight-nucleate) stages of development. The presence of supernumerary nuclei is considered to be a consequence of the production of coenocytic megaspores. The production of 2n eggs was confirmed through cytological investigation by means of the diameter of the egg-cell nucleolus. The frequency of 2n eggs was lower than the frequency of binucleated macrospores as previously determined. This discrepancy may be due to environmental effects but also to the fact that binucleated macrospores may degenerate or may, after two mitotic divisions, give rise to eight-nucleated embryo sacs counted as normals.     

Cytological,morphological and molecular analyses of controlled progenies from meiotic mutants of alfalfa producing unreduced gametes

A program of sexual polyploidization was carried out in alfalfa using plants from wild diploid species that produced male or female unreduced gametes. Sixteen progenies from 2x-4x and 2x-2x crosses were examined with a combination of morphological, cytological and molecular analyses. The chromosome counts revealed diploid, tetraploid and aneuploid plants. Plants with B chromosomes were also detected. The leaf area of the plants was a useful characteristic for distinguishing tetraploid from diploid plants obtained by unilateral or bilateral sexual polyploidization. Leaf shape and leaf margin were not correlated with the ploidy levels. Plants with supernumerary chromosomes displayed obovate or elliptic leaves which differed markedly from the range of forms typical of diploid and tetraploid alfalfa plants. RAPD markers were investigated in all progeny plants to determine maternal and paternal amplification products. Three alfalfa-specific primers proved to be effective in revealing the hybrid origin of the plants. A combination of cytological, morphological and molecular analyses is essential for a detailed genetic characterization of progenies in programs of sexual polyploidization.     

Theoretical value of estimates of general combining ability in the autotetraploid crop

Summary The means of half-sib progenies have been indicated as selection criteria for intra-population improvement while the average of the means of full-sib progenies in diallel analyses have been proposed as predictors, in part, of the means of untested synthetic varieties. When these measures based on progeny means are expressed as deviations from a defined greater population of crosses, they are often termed the general combining ability (GCA). In this study the GCA estimates or a facsimile were theoretically investigated for the one locus, digene, autotetraploid model to verify the genetic basis and its value for selection and prediction in the presence of a naturally occurring phenomena of autopolyploids called gametic disequilibrium with three types of non-additive inheritance. Two breeding objectives were envisioned, the selection of best parents with recurrent selection based on GCA in the continued development of elite populations and the prediction of advanced generation synthetic variety performance. The first generation means of progenies with a potential bias due to gametic disequilibrium were compared to GCA estimation of same progenies in the absence of gametic disequilibrium. The results indicated that testcrossing plants to a population without gametic disequilibrium could be used for selection of best parents. The gametic disequilibrium in the cross may increase or depress selection response dependent on the array of genotypes which happen to be evaluated, on the type of genic action at the locus, and on the frequency of the desirable allele in the testor population. The GCA estimates for prediction of synthetic performance were potentially biased by gametic disequilibrium. An assumption of pollination by the same array of gametes was made for all plants, but obviously was unrealistic for GCA estimation with partial diallels, or with no selfing, and in other situations. The GCA estimate was shown to be an unreliable predictor of synthetic variety performance. When it was assumed that different plants were pollinated by different arrays of gametes, a more realistic situation, no genetic interpretation of GCA values was possible even with purely additive gene action at the locus.Cooperative investigation of the Alfalfa Production Research Unit, United States Department of Agriculture, Agricultural Research Service, and the Nevada Agricultural Experiment Station, Reno, Nevada     

Photosynthate partitioning into leaf starch as affected by daily photosynthetic period duration in six species

Starch accumulation rates increased up to five-fold in the leaves of five out of six species examined when plants were shifted from a long (12 or 14-h) to a short (7-h) photosynthetic period. The five species that responded were corn (Zea mays L.), pangola (Digitaria decumbens Stent.), soybean (Glycine max L.) Merr.), spinach (Spinacia oleracea L.), and sugarbeet (Beet vulgaris L.) However, the rate of starch accumulation in alfalfa (Medicago sativa L.) was unaltered following a shift from a long to a short photosynthetic period. We conclude that photosynthate partitioning into chloroplast starch, at least in a wide variety of species, is a programmable process that can be manipulated by altering the length of the daily photosynthetic period.     

Effect of gametic disequilibrium on means and on genetic variances of autotetraploid synthetic varieties

Summary The occurrence and effects of a gametic disequilibrium (DSE) in the first generation of a theoretical two-population synthetic variety were investigated. Theoretical development was limited to the genetics at a single locus with two alleles in an autotetraploid species with random chromosome inheritance. Algebraic expressions were developed for the differences between the mean genotypic values of the two-population synthetic variety at generation one and in random mating equilibrium (RME). For the situation where both parents of the synthetic were in RME, a numerical analysis was performed for all possible allele frequencies assuming the following types of genic action: monoplex dominance, partial monoplex dominance, duplex dominance, partial duplex dominance, and additive. The result indicated that with non-additive genic action the DSE could, in some cases, greatly depress or inflate the mean genotypic value of the first generation (Syn-1_(RME)). Thus, any change of means over advancing generations with loss of DSE could be positive or negative. When additive genic action was assumed, there was no effect associated with DSE and when both parents had the same allele frequencies there was no DSE. The DSE, with only a minor exception, decreased the genetic variance and in numerous cases forced it near zero. Expressions were developed for mean genotypic values of a first generation synthetic with DSE in one parent (Syn-1_(DSE/RME)) or both parents (Syn-1_(DSE)). The deviation of these means from those of Syn-1_(RME) was a function of digenic and quadragenic population effects. An inspection of the response equations for Syn-1_(RME) indicated that in a series of crosses with one common parent the rankings of first generation means would be the same as the ranking of populations at equilibrium though the individual means would be biased. More importantly with DSE of one or both parents there are situations when a ranking of first generation mean genotypic values would not reflect relative frequency of desirable alleles in the populations. These results indicate that statistical analyses and selections based on means of the Syn-1 generation can have an error which is not avoidable by improvement in precision of evaluation.Cooperative investigations of the Alfalfa Production Research Unit, United States Department of Agriculture, Agricultural Research Service, and the Nevada Agricultural Experiment Station, Reno, Nevada, USAPaper No. 590 Scientific Journal Series, Nevada Agricultural Experiment Station     

Biparental inheritance of chloroplast DNA and the existence of heteroplasmic cells in alfalfa

Summary Mapping of chloroplast DNA (ctDNA) restriction fragment patterns from a chlorophyll deficient mutant and two phenotypically normal alfalfa genotypes (Medicago sativa L.) has demonstrated the existence of a distinct ctDNA genotype from each source. These unique restriction fragment patterns were utilized to identify maternal or paternal origin of ctDNA in hybrid plants from crosses involving the normal alfalfa genotypes as females and the yellow-green chlorophyll deficient sectors as males. Progeny from these crosses expressing the yellow-green sectored phenotypes contained paternal ctDNA in the chlorophyll deficient sectors and maternal ctDNA in the normal sectors, confirming biparental plastid inheritance. The existence of mixed cells containing both mutant and normal plastids at various stages of sorting-out was observed by transmission electron microscopy of mesophyll cells in mosaic tissue from hybrid plants. This observation verified the biparental transmission of plastids in alfalfa.     

Observations on the mating behavior of the alfalfa blotch leafminer,Agromyza frontella (Rondani) (Diptera: Agromyzidae), and evidence of a female sex pheromone

The mating behavior of Agromyza frontella was studied under laboratory conditions. Adults were able to mate on the day of emergence, with no evident periodicity throughout the photophase. The host plant was essential for mating to occur, its presence affecting female receptivity rather than male copulatory behavior. Males generally entered a stationary phase once in the proximity of a female, before undertaking a final approach. This stationary behavior frequently resulted in male aggregations around a female, and under such conditions males exhibited a characteristic wing vibrating behavior. As male wing vibration was not an essential behavior for successful mating, and rarely occurred during male encounters in the absence of females or when only one male was near a female, it was considered as being primarily a male-male signal. The majority of females that mated exhibited an ovipositor pumping behavior that stimulated the male approach. However, such behavior was not essential to attract mates, as dead females elicited the entire sequence of male mating behavior. This suggested the presence of a cuticular sex pheromone, as reported for other species of higher Diptera. Whole virgin female (<24- h or 3- day-old) hexane extracts applied to male cadavers increased the time males spent on the plant, the number of contacts with the treated cadavers, the incidence of attempted copulations, and the wing vibrating behavior between males compared with controls using untreated cadavers. The results obtained indicate that females control copulation in A. frontella and that both semiochemical and visual cues are important in eliciting male mating behavior.     

The influence of addition of gallic acid, tannic acid, or quebracho tannins to alfalfa hay on in vitro rumen fermentation and microbial protein synthesis

Tannins may reduce rumen degradability of protein, increase the proportion of feed protein reaching the lower digestive tract for enzymatic digestion and thereby increase the efficiency of protein utilization. The objective was to assess the effects of different types and levels of tannins on rumen in vitro gas production and its kinetics, in vitro true degradability (IVTD) and rumen degradability of protein (IVRDP), and microbial protein synthesis by incubating alfalfa (Medicago sativa L.) hay in buffered rumen fluid. Alfalfa was incubated in buffered rumen fluid with and without the addition of different levels of gallic acid (GA), quebracho tannin (QT), or tannic acid (TA). Tannins at the lower inclusion levels had minimal effects on fermentation products compared to the higher levels. Addition of QT and TA reduced ammonium-N (NH₄⁺-N) concentration. Addition of QT at 20, 40, and 60 g/kg DM decreased NH₄⁺-N by 2, 7, and 12% compared with control whereas addition of TA reduced NH₄⁺-N by 5, 6, and 12% when added at 20, 40 and 60 g/kg DM, respectively. In experiment 2, addition of QT at 50, 100, and 150 g/kg DM, resulted in reduction of NH₄⁺-N by 12, 30, and 51%, respectively, compared with the control. Addition of TA at 50, 100, and 150 g/kd DM reduced NH₄⁺-N by 14, 26, and 47% compared with control. Inclusion of QT at 50, 100, 150 DM reduced IVRDP by 13, 30, and 36% compared with control whereas at these levels of inclusion, TA resulted in reduction of IVRDP by 14, 25, and 48%. Rate of gas production decreased (P<0.001), while asymptotic gas production increased (P<0.0001) with increasing level of GA and TA. Quebracho tannin decreased (P<0.0001) both the rate and asymptotic of gas production. Gallic acid had a positive effect on fermentation as indicated by increased gas production and total short chain fatty acids (SCFA) production. Quebracho tannin decreased 24 h gas production, IVTD, and total SCFA production. Acetate to propionate ratio increased with the addition of GA and but decreased when QT was added. Addition of tannins did not markedly increase total purines but numerical values tended to be higher in the presence of tannins compared with the control. Efficiency of microbial growth was lower in the presence of GA and unaltered by TA, but higher in the presence of QT compared with the control. The effect of tannins on rumen fermentation and protein degradation varied with type and level of tannins. In vivo studies will be conducted to validate the in vitro results.     

Effect of proteinase inhibitors on Indian alfalfa weevil (<Emphasis Type="Italic">Hypera postica</Emphasis> Gyll.) growth and development

Three families of proteinase inhibitors, namely, serine, cysteine (thiol) and aspartic (carboxyl) were examined for their inhibitory effects on growth and development of Indian alfalfa weevil (Coleoptera: Curculionidae). Proteinase inhibitors are considered as a part of alternate strategy to control the herbivorous insect as they inhibit the digestive enzymes of the insects. Larval leaf feeding, survival, pupation and adult emergence were significantly decreased by pHMB, (p-hydroxy-mercuribenzoic acid), cystatin and E-64 (trans-epoxysuccinyl-l-leucylamido-(4guanidino)-butane) belonging to cysteine class of proteinases, at a concentration of 0.1 and 0.5%. Serine and aspartic classes of inhibitors have low detrimental effects on larvae. The results demonstrate the inhibitory response of specific proteinase inhibitors on alfalfa weevil larval leaf feeding, survival, pupation and adult emergence. Weevil resistant species, namely, Medicago scutellata showed high level of leaf consumption under forced feeding in vivo bioassay indicated the presence of resistance factors other than proteinase inhibitors.