Genome wide changes in gene expression were monitored in the drought tolerant C4 cereal Sorghum bicolor, following exposure of seedlings to high salinity (150 mM NaCl), osmotic stress (20% polyethylene glycol) or abscisic acid (125 microM ABA). A sorghum cDNA microarray providing data on 12,982 unique gene clusters was used to examine gene expression in roots and shoots at 3- and 27-h post-treatment. Expression of approximately 2200 genes, including 174 genes with currently unknown functions, of which a subset appear unique to monocots and/or sorghum, was altered in response to dehydration, high salinity or ABA. The modulated sorghum genes had homology to proteins involved in regulation, growth, transport, membrane/protein turnover/repair, metabolism, dehydration protection, reactive oxygen scavenging, and plant defense. Real-time PCR was used to quantify changes in relative mRNA abundance for 333 genes that responded to ABA, NaCl or osmotic stress. Osmotic stress inducible sorghum genes identified for the first time included a beta-expansin expressed in shoots, actin depolymerization factor, inositol-3-phosphate synthase, a non-C4 NADP-malic enzyme, oleosin, and three genes homologous to 9-cis-epoxycarotenoid dioxygenase that may be involved in ABA biosynthesis. Analysis of response profiles demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to ABA, high salinity and water deficit. Modulation of genes involved in signal transduction, chromatin structure, transcription, translation and RNA metabolism contributes to sorghum's overlapping but nonetheless distinct responses to ABA, high salinity, and osmotic stress. Overall, this study provides a foundation of information on sorghum's osmotic stress responsive gene complement that will accelerate follow up biochemical, QTL and comparative studies.

The Ma, gene is one of six genes that regulate the photoperiodic sensitivity of flowering in sorghum (Sorghum bicolor [L.] Moench).The masR mutation of this gene causes a phenotype that is similar to plants that are known to lack phytochrome B, and masR sorghum lacks a 123-kD phytochrome that predominates in light-grown plants and that is present in non-magR plants. A population segregating for Ma, and masR was created and used to identify two randomly amplified polymorphic DNA markers linked to Ma,. These two markers were cloned and mapped in a recombinant inbred population as restriction fragment length polymorphisms. cDNA clones of PHYA and PHYC were cloned and sequenced from a cDNA library prepared from green sorghum leaves. Using a genome-walking technique, a 7941 -bp partia1 sequence of PHYB was determined from genomic DNA from masR sorghum. PHYA, PHYB, and P H Y C all mapped to the same linkage group. The Ma,-linked markers mapped with PHYB more than 121 centimorgans from PHYA and PHYC. A frameshift mutation resulting in a premature stop codon was found in the PHYB sequence from magR sorghum. Therefore, we conclude that the Ma, locus in sorghum is a PHYB gene that encodes a 123-kD phytochrome.The transition from vegetative to reproductive growth is the result of the activation of genes responsible for inflorescence and floral organ formation. These genes, which control apex identity and floral organ morphogenesis, are strictly regulated, since their improper expression results in abnormal flowers and inflorescences (Okamuro et al., 1993;Veit et al., 1993). The initial activation of these genes is usually the result of environmental cues that indicate an appropriate time to flower. The mechanisms by which environmental factors activate inflorescence and floral organ production are complex and many genes are known to be involved in the transduction of environmental signals that regulate flowering (Bernier et al., 1993; Coupland, 1995).Of all of the environmental factors that are sensed by plants, daylength is probably the most important in inducing flowering. The phenomenon whereby daylength regulates flowering is referred to as photoperiodism. An understanding of the effect of daylength on reproductive development has agronomic importance because the ability to alter flowering time allows the cultivation of a species in environments that differ greatly from the one in which it originally evolved. Our understanding of photoperiodism has historically relied upon a physiological examination of the phenomenon. Recently, genetic analysis of floral induction has provided new insights into this process. In the LD plant Arabidopsis thaliana a series of genes has been recognized that influences flowering time, and these genes have been categorized into six phenotypic groups based on earliness or lateness in flowering in response to short days, long days, and vernalization; (Coupland, 1995). The existence of these separate phenotypic classes suggests the existence of severa1 pathways that regulate photoperiod sens...

The phytochrome photoreceptors play important roles in the photoperiodic control of vegetative bud set, growth cessation, dormancy induction, and cold-hardiness in trees. Interestingly, ecotypic differences in photoperiodic responses are observed in many temperate-zone tree species. Northern and southern ecotypes of black cottonwood (Populus trichocarpa Torr. & Gray), for example, exhibit marked differences in the timing of short-day-induced bud set and growth cessation, and these responses are controlled by phytochrome. Therefore, as a first step toward determining the molecular genetic basis of photoperiodic ecotypes in trees, we characterized the phytochrome gene (PHY) family in black cottonwood. We recovered fragments of one PHYA and two PHYB using PCR-based cloning and by screening a genomic library. Results from Southern analyses confirmed that black cottonwood has one PHYA locus and two PHYB loci, which we arbitrarily designated PHYB1 and PHYB2. Phylogenetic analyses which included PHY from black cottonwood, Arabidopsis thaliana and tomato (Solanum lycopersicum) suggest that the PHYB/D duplications in these species occurred independently. When Southern blots were probed with PHYC, PHYE, and PHYE heterologous probes, the strongest bands that we detected were those of black cottonwood PHYA and/or PHYB. These results suggest that black cottonwood lacks members of the PHYC/F and PHYE subfamilies. Although black cottonwood could contain additional PHY that are distantly related to known angiosperm PHY, our results imply that the PHY family of black cottonwood is less complex than that of other well-characterized dicot species such as Arabidopsis and tomato. Based on Southern analyses of five black cottonwood genotypes representing three photoperiodic ecotypes, substantial polymorphism was detected for at least one of the PHYB loci but not for the PHYA locus. The novel character of the PHY family in black cottonwood, as well as the differences in polymorphism we observed between the PHYA and PHYB subfamilies, indicates that a number of fundamental macro- and microevolutionary questions remain to be answered about the PHY family in dicots.

Improved knowledge of the sorghum transcriptome will enhance basic understanding of how plants respond to stresses and serve as a source of genes of value to agriculture. Toward this goal, Sorghum bicolor L. Moench cDNA libraries were prepared from light-and dark-grown seedlings, drought-stressed plants, Colletotrichum-infected seedlings and plants, ovaries, embryos, and immature panicles. Other libraries were prepared with meristems from Sorghum propinquum (Kunth) Hitchc. that had been photoperiodically induced to flower, and with rhizomes from S. propinquum and johnsongrass (Sorghum halepense L. Pers.). A total of 117,682 expressed sequence tags (ESTs) were obtained representing both 3# and 5# sequences from about half that number of cDNA clones. A total of 16,801 unique transcripts, representing tentative UniScripts (TUs), were identified from 55,783 3# ESTs. Of these TUs, 9,032 are represented by two or more ESTs. Collectively, these libraries were predicted to contain a total of approximately 31,000 TUs. Individual libraries, however, were predicted to contain no more than about 6,000 to 9,000, with the exception of light-grown seedlings, which yielded an estimate of close to 13,000. In addition, each library exhibits about the same level of complexity with respect to both the number of TUs preferentially expressed in that library and the frequency with which two or more ESTs is found in only that library. These results indicate that the sorghum genome is expressed in highly selective fashion in the individual organs and in response to the environmental conditions surveyed here. Close to 2,000 differentially expressed TUs were identified among the cDNA libraries examined, of which 775 were differentially expressed at a confidence level of 98%. From these 775 TUs, signature genes were identified defining drought, Colletotrichum infection, skotomorphogenesis (etiolation), ovary, immature panicle, and embryo.