Chalcone synthase, a key regulatory enzyme in the flavonoid pathway, constitutes an eight-member gene family in Glycine max (soybean). Three of the chalcone synthase (CHS) gene family members are arranged as inverted repeats in a 10-kb region, corresponding to the I locus (inhibitor). Spontaneous mutations of a dominant allele (I or i i ) to a recessive allele (i) have been shown to delete promoter sequences, paradoxically increasing total CHS transcript levels and resulting in black seed coats. However, it is not known which of the gene family members contribute toward pigmentation and how this locus affects CHS expression in other tissues. We investigated the unusual nature of the I locus using four pairs of isogenic lines differing with respect to alleles of the I locus. RNA gel blots using a generic open reading frame CHS probe detected similar CHS transcript levels in stems, roots, leaves, young pods, and cotyledons of the yellow and black isolines but not in the seed coats, which is consistent with the dominant I and i i alleles mediating CHS gene silencing in a tissue-specific manner. Using real-time RT-PCR, a variable pattern of expression of CHS genes in different tissues was demonstrated. However, increase in pigmentation in the black seed coats was associated with release of the silencing effect specifically on CHS7/CHS8, which occurred at all stages of seed coat development. These expression changes were linked to structural changes taking place at the I locus, shown to encompass a much wider region of at least 27 kb, comprising two identical 10.91-kb stretches of CHS gene duplications. The suppressive effect of this 27-kb I locus in a specific tissue of the G. max plant represents a unique endogenous gene silencing mechanism.

Transcript profiling during susceptible (S) and hypersensitive response-associated resistance (R) interactions was determined in soybean (Glycine max). Pseudomonas syringae pv. glycinea carrying or lacking the avirulence gene avrB, was infiltrated into cultivar Williams 82. Leaf RNA was sampled at 2, 8, and 24 h postinoculation (hpi). Significant changes in transcript abundance were observed for 3,897 genes during the experiment at P < or = 0.000005. Many of the genes showed a similar direction of increase or decrease in abundance in both the S and R responses, but the R response generally showed a significantly greater degree of differential expression. More than 25% of these responsive genes had not been previously reported as being associated with pathogen interactions, as 704 had no functional annotation and 378 had no homolog in National Center for Biotechnology Information databases. The highest number of transcriptional changes was noted at 8 hpi, including the downregulation of 94 chloroplast-associated genes specific to the R response. Photosynthetic measurements were consistent with an R-specific reduction in photosystem II operating efficiency (phiPSII) that was apparent at 8 hpi for the R response with little effect in the S or control treatments. Imaging analyses suggest that the decreased phiPSII was a result of physical damage to PSII reaction centers.

Seed coat color in soybean is determined by four alleles of the classically defined I (inhibitor) locus that controls the presence or absence as well as the spatial distribution of anthocyanin pigments in the seed coat. By analyzing spontaneous mutations of the I locus, we demonstrated that the I locus is a region of chalcone synthase (CHS) gene dupllcations. Paradoxically, deletions of CHS gene sequences allow higher levels of CHS mRNAs and restore pigmentation to the seed coat. The unusual nature of the I locus suggests that its dominant alleles may represent naturally occurring examples of homology-dependent gene silenclng and that the spontaneous deletions erase the gene-silencing phenomena. Specifically, mutations from the dominant i' allele (yellow seed coats wlth pigmented hila) to the recessive i allele (fully pigmented) can be associated wlth the absence of a 2.3-W Hindlll fragment that carries CHS4, a member of the multigene CHS family. Seven independent mutatlons exhibit deletions in the CHS4 promoter mgion. The dominant I allele (yellow seed coats) exhibits an extra 12.1-kb Hindlll fragment that hybridlzes with both the CHS coding region and CHSí promoter-specific probes. Mutatlons of the dominant I allele to the mcessive i allele (pigmented seed coats) give rise to 10.4-or 9.6-kb Hindlll CHS fragments that have lost the duplicated CHSí promoter. Finally, gene expression analysis demonstrated that heterozygous plants (//i) wlth yellow seed coats have reduced mRNA levels, indlcatlng that the 12.1-kb Hindlll CHS fragment associated with the dominant I allele inhibits pigmentation in a trans-dominant manner. Moreover, CHS gene-specific expression in seed coats shows that multiple CHS genes are expressed ln seed coats.

Globular somatic embryos can be induced from immature cotyledons of soybean (Glycine max L. Merr. cv Jack) placed on high levels of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos develop from the adaxial side of the cotyledon, whereas the abaxial side evolves into a callus. Using a 9,280-cDNA clone array, we have compared steady-state RNA from the adaxial side from which embryos develop and from the abaxial callus at five time points over the course of the 4 weeks necessary for the development of globular embryos. In a second set of experiments, we have profiled the expression of each clone in the adaxial side during the same period. A total of 495 genes differentially expressed in at least one of these experiments were grouped according to the similarity of their expression profiles using a nonhierarchical clustering algorithm. Our results indicate that the appearance of somatic embryos is preceded by dedifferentiation of the cotyledon during the first 2 weeks on auxin. Changes in mRNA abundance of genes characteristic of oxidative stress and genes indicative of cell division in the adaxial side of the cotyledons suggest that the arrangement of the new cells into organized structures might depend on a genetically controlled balance between cell proliferation and cell death. Our data also suggest that the formation of somatic globular embryos is accompanied by the transcription of storage proteins and the synthesis of gibberellic acid.Due to their ability to regenerate into full plants, somatic embryos are the tissue of choice for transformation by particle bombardment in several crop species including soybean (Glycine max L. Merr. cv Jack; Finer and McMullen, 1991). In soybean, somatic embryos are obtained by induction, or culturing, of immature cotyledons on high concentration of auxin. Globular somatic embryos originate from epidermal and subepidermal cells of the adaxial side of the cotyledon (Finer, 1988), whereas the abaxial side evolves into a callus. The adaxial side is the "flat" side of the cotyledons, closest to the axis of the embryo, whereas the abaxial side is the side of the cotyledon in contact with the endosperm and the seed coat. However, the response to tissue culture is highly genotype dependent (Meurer et al., 2001), and the ability to transform a wider range of cultivars could accelerate the production of transgenic plants.Somatic and zygotic embryos follow the same general pattern of development (Zimmerman, 1993;Goldberg et al., 1994). However, large quantities of somatic embryos can be produced in vitro, making them more amenable to experimentation than their zygotic counterparts, which are protected by fruit structures and less accessible. Therefore, somatic embryos constitute a model system to study basic aspects of embryogenesis, as well as a tool for efficient transformation.Little is known of the genes expressed in early globular stage embryos (Zimmerman, 1993). Choi evaluated that only 10% of the proteins visible on a two-dimensional gel are embryo specific (Choi ...

Two dominant alleles of the I locus in Glycine max silence nine chalcone synthase (CHS) genes to inhibit function of the flavonoid pathway in the seed coat. We describe here the intricacies of this naturally occurring silencing mechanism based on results from small RNA gel blots and high-throughput sequencing of small RNA populations. The two dominant alleles of the I locus encompass a 27-kb region containing two perfectly repeated and inverted clusters of three chalcone synthase genes (CHS1, CHS3, and CHS4). This structure silences the expression of all CHS genes, including CHS7 and CHS8, located on other chromosomes. The CHS short interfering RNAs (siRNAs) sequenced support a mechanism by which RNAs transcribed from the CHS inverted repeat form aberrant double-stranded RNAs that become substrates for dicer-like ribonuclease. The resulting primary siRNAs become guides that target the mRNAs of the nonlinked, highly expressed CHS7 and CHS8 genes, followed by subsequent amplification of CHS7 and CHS8 secondary siRNAs by RNA-dependent RNA polymerase. Most remarkably, this silencing mechanism occurs only in one tissue, the seed coat, as shown by the lack of CHS siRNAs in cotyledons and vegetative tissues. Thus, production of the trigger double-stranded RNA that initiates the process occurs in a specific tissue and represents an example of naturally occurring inhibition of a metabolic pathway by siRNAs in one tissue while allowing expression of the pathway and synthesis of valuable secondary metabolites in all other organs/tissues of the plant.