Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean

Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KQEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.     

Applications of minirhizotrons to understand root function in forests and other natural ecosystems

Minirhizotrons have proved useful to understand the dynamics and function of fine roots. However, they have been used comparatively infrequently in forests and other natural plant communities. Several factors have contributed to this situation, including anomalous root distributions along the minirhizotron surface and the difficulty of extracting data from minirhizotron images. Technical and methodological advances have ameliorated some of these difficulties, and minirhizotrons have considerable potential to address some questions of long standing interest. These questions include more fully understanding the role of roots in carbon and nutrient cycling, rates of root decomposition, responses to resource availability and the functional significance of interactions between plant roots and soil organisms. Maximizing the potential for minirhizotrons to help us better understand the functional importance of fine roots in natural plant communities depends upon using them to answer only those questions appropriate to both their inherent strengths and limitations.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Markers for selection of the rice Xa21 disease resistance gene

Six molecular markers were mapped to a 7.4-cM region of rice chromosome 11 containing the Xa21 gene, which confers resistance to the pathogen Xanthomonas oryzae pv oryzae. Three markers, RG103, 248 and 818, co-segregated with Xa21 in a population of 1141 plants. Multiple copies of all marker loci were present within the region that was introgressed from Oryza longistaminata into O. sativa. The marker loci were cloned and primers were designed that defined sequence-tagged sites. Physical mapping of the three tightly linked central markers revealed that RG103, the marker that hybridizes to the Xa21 gene, resides on a separate DNA fragment from the other two markers.Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Characterization of Mhc genes in a multigenerational family of ring-necked pheasants

Little is known about the major histocompatibility (Mhc) genes of birds in different taxonomic groups or about how Mhc genes may be organized in avian species divergent by evolution or habitat. Yet it seems likely that much might be learned from birds about the evolution, organization, and function of this intricate complex of polymorphic genes. In this study a close relative of the chicken, the ring-necked pheasant (Phasianus colchicus), was examined for the presence and organization of Mhc B-G genes. The patterns of restriction fragments revealed by chicken B-G probes in Southern hybridizations and the patterns of pheasant erythrocyte polypeptides revealed in immunoblots by antisera raised against chicken B-G polypeptides provide genetic, molecular, and biochemical data confirming earlier serological evidence for the presence of B-G genes in the pheasant, and hence, the presence of a family of B-G genes in at least a second species of birds. The high polymorphism exhibited by the pheasant B-G gene family allowed genetic differences among individuals within the small experimental population in this study to be detected easily by restriction fragment patterns. Further evidence was found for the organization of the pheasant Mhc class I and class II genes into genetically independent clusters. Whether these gene clusters are fully comparable to the B and Rfp-Y systems in the chicken or whether yet another organization of Mhc genes has been encountered in the pheasant remains to be determined.     

Effects ofPax3Modifier Genes on Craniofacial Morphology, Pigmentation, and Viability: A Murine Model of Waardenburg Syndrome Variation

Waardenburg syndrome type 1 is caused by mutations inPAX3.Over 50 humanPAX3mutations that lead to hearing, craniofacial, limb, and pigmentation anomalies have been identified. APAX3mutant allele, segregating in a family, can show reduced penetrance and variable expressivity that cannot be explained by the nature of the mutation alone. TheMus musculus Pax3mutationSp^d(Splotch-delayed, Pax3[formula]), coisogenic on the C57BL/6J (B₆) genetic background, produces in heterozygotes a white belly spot with 100% penetrance and very few other anomalies. By contrast, manySp^d/+ BC₁progeny [F₁♀Sp^d/+ (♀Sp^d/+ B₆× ♂ +/+Mus spretus) × ♂ +/+ B₆] exhibit highly variable craniofacial and pigmentary anomalies. Of the BC₁Sp^d/+ progeny, 23.9% are estimated to be nonviable, and 32.1% are nonpenetrant for the white belly spot. The penetrance and expressivity of theSp^d/+ genotype are controlled in part by the genetic background and the sex of the individual. A minimum of two genes interact withSp^dto influence the craniofacial features of these mice. One of these genes may be either X-linked or sex-influenced, while the other is autosomal. TheA-locus (Agouti) or a gene closely linked toAalso plays a role in determining craniofacial features. At least one additional gene, possibly theA-locus or a gene linked toA,interacts withSp^dand determines the presence and size of the white belly spot. The viability of BC₁mice is influenced by at least three factors:Sp^d,A-locus alleles or a gene closely linked to theA-locus, and the sex of the mouse. These BC₁mice provide an opportunity to identify genes that interact with and modify the expression ofPax3and serve as a model to identify the genes that modify the expression of humanPAX3mutations.     

Localization of mating behavior of releasedBactrocera dorsalis flies on host fruit in an orchard

Oriental fruit flies,Bactrocera dorsalis, cultured in the laboratory for six generations, were released when 12–14 days old in an orchard of nonfruiting host trees that were furnished with either food and water, nonpunctured host fruit, punctured host fruit, or no resources. Nearly all flies of both sexes, all sexual behavior of males, and all mating pairs were observed on trees with fruit. Moreover, on trees with fruit, nearly all flies of both sexes, nearly all sexual behavior of males, and nearly all mating pairs occurred on the fruit itself rather than on the foliage or branches. In a subsequent test, both sexes were found to be strongly attracted to the odor of host fruit. For a polyphagous species of a tropical tephritid, these findings are the first to show a high level of male aggregation and a high amount of male sexual behavior on the fruit of host trees. Findings are discussed in relation to current knowledge of tephritid mating behavior. An additional quality-control test for laboratory cultured males used in the sterile insect technique of fly management is recommended.     

Projection pattern of sensory neurons in the central nervous system of a homeotic mutation of the moth Manduca sexta

Octopod (Octo) is a mutation of the moth Manduca sexta, which transforms the first abdominal segment (A1) in the anterior direction. Mutant animals are characterized by the appearance of homeotic thoracic-like legs on A1. We exploited this mutation to determine what rules might be used in specifying the fates of sensory neurons located on the body surface of larval Manduca. Mechanical stimulation of homeotic leg sensilla did not cause reflexive movements of the homeotic legs, but elicited responses similar to those observed following stimulation of ventral A1 body wall hairs. Intracellular recordings demonstrated that several of the motoneurons in the A1 ganglion received inputs from the homeotic sensory hairs. The responses of these motoneurons to stimulation of homeotic sensilla resembled their responses to stimulation of ventral body wall sensilla. Cobalt fills revealed that the mutation transformed the segmental projection pattern of only the sensory neurons located on the ventral surface of A1, resulting in a greater number with intersegmental projection patterns typical of sensory neurons found on the thoracic body wall. Many of the sensory neurons on the homeotic legs had intersegmental projection patterns typical of abdominal sensory neurons: an anteriorly directed projection terminating in the third thoracic ganglion (T3). Once this projection reached T3, however, it mimicked the projections of the thoracic leg sensory neurons. These results demonstrate that the same rules are not used in the establishment of the intersegmental and leg-specific projection patterns. Segmental identity influences the intersegmental projection pattern of the sensory neurons of Manduca, whereas the leg-specific projections are consistent with a role for positional information in determining their pattern. © 1995 John Wiley & Sons, Inc.     

Heartbeat control in the medicinal leech: A model system for understanding the origin,coordination, and modulation of rhythmic motor patterns

We have analyzed in detail the neuronal network that generates heartbeat in the leech. Reciprocally inhibitory pairs of heart interneurons form oscillators that pace the heartbeat rhythm. Other heart interneurons coordinate these oscillators. These coordinating interneurons, along with the oscillator interneurons, form an eight-cell timing oscillator network for heartbeat. Still other interneurons, along with the oscillator interneurons, inhibit heart motor neurons, sculpting their activity into rhythmic bursts. Critical switch interneurons interface between the oscillator interneurons and the other premotor interneurons to produce two alternating coordination states of the motor neurons. The periods of the oscillator interneurons are modulated by endogenous RFamide neuropeptides. We have explored the ionic currents and graded and spike-mediated synaptic transmission that promote oscillation in the oscillator interneurons and have incorporated these data into a conductance-based computer model. This model has been of considerable predictive value and has led to new insights into how reciprocally inhibitory neurons produce oscillation. We are now in a strong position to expand this model upward, to encompass the entire heartbeat network, horizontally, to elucidate the mechanisms of FMRFamide modulation, and downward, to incorporate cellular morphology. By studying the mechanisms of motor pattern formation in the leech, using modeling studies in conjunction with parallel physiological experiments, we can contribute to a deeper understanding of how rhythmic motor acts are generated, coordinated, modulated, and reconfigured at the level of networks, cells, ionic currents, and synapses. © 1995 John Wiley & Sons, Inc.