The effect of sulfur nutrition on plant glucosinolate content: physiology and molecular mechanisms.

Glucosinolates are sulfur-rich plant metabolites of the order Brassicales that function in the defense of plants against pests and pathogens. They are also important in human society as flavor components, cancer-prevention agents, and crop biofumigants. Since glucosinolates may represent up to 30 % of the total sulfur content of plant organs, their accumulation should depend intimately on the sulfur status of the entire plant. Here we review the literature on how sulfur supply affects glucosinolate content. In field and greenhouse experiments involving soil, hydroponic and tissue culture media, sulfur fertilisation usually led to an increase in glucosinolate content ranging from 25 % to more than 50-fold, depending on the plant species, amount of sulfur applied, and type of treatment. The effect was greater on glucosinolates derived from the sulfur amino acid, methionine, than on glucosinolates derived from tryptophan. These changes are regulated not by simple mass action effects, but by extensive changes in gene transcription. In sulfur-deficient plants, there is a general down-regulation of glucosinolate biosynthetic genes which accompanies an up-regulation of genes controlling sulfur uptake and assimilation. Glucosinolates may be considered a potential source of sulfur for other metabolic processes under low-sulfur conditions, since increased breakdown of glucosinolates has been reported under sulfur deficiency. However, the pathway for sulfur mobilisation from glucosinolates has not been determined. The breakdown of indolic glucosinolates to form auxin in roots under sulfur-deficient conditions may help stimulate root formation for sulfur uptake.

A gene controlling variation in Arabidopsis glucosinolate composition is part of the methionine chain elongation pathway.

Arabidopsis and other Brassicaceae produce an enormous diversity of aliphatic glucosinolates, a group of methionine (Met)-derived plant secondary compounds containing a beta-thio-glucose moiety, a sulfonated oxime, and a variable side chain. We fine-scale mapped GSL-ELONG, a locus controlling variation in the side-chain length of aliphatic glucosinolates. Within this locus, a polymorphic gene was identified that determines whether Met is extended predominantly by either one or by two methylene groups to produce aliphatic glucosinolates with either three- or four-carbon side chains. Two allelic mutants deficient in four-carbon side-chain glucosinolates were shown to contain independent missense mutations within this gene. In cell-free enzyme assays, a heterologously expressed cDNA from this locus was capable of condensing 2-oxo-4-methylthiobutanoic acid with acetyl-coenzyme A, the initial reaction in Met chain elongation. The gene methylthioalkylmalate synthase1 (MAM1) is a member of a gene family sharing approximately 60% amino acid sequence similarity with 2-isopropylmalate synthase, an enzyme of leucine biosynthesis that condenses 2-oxo-3-methylbutanoate with acetyl-coenzyme A.

Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase.

Poikilothermic organisms require mechanisms that allow survival at chilling temperatures (2 to 15 degreesC). We have isolated chilling-sensitive mutants of Arabidopsis, a plant that is very chilling resistant, and are characterizing them to understand the genes involved in chilling resistance. The T-DNA-tagged mutant paleface1 (pfc1) grows normally at 22 degrees C but at 5 degrees C exhibits a pattern of chilling-induced chlorosis consistent with a disruption of chloroplast development. Genomic DNA flanking the T-DNA was cloned and used to isolate wild-type genomic and cDNA clones. The PFC1 transcript is present at a low level in wild-type plants and was not detected in pfc1 plants. Wild-type Arabidopsis expressing antisense constructs of PFC1 grew normally at 22 degrees C but showed chilling-induced chlorosis, confirming that the gene is essential for low-temperature development of chloroplasts. The deduced amino acid sequence of PFC1 has identity with rRNA methylases found in bacteria and yeast that modify specific adenosines of pre-rRNA transcripts. The pfc1 mutant does not have these modifications in the small subunit rRNA of the plastid.

Does the ocs-element occur as a functional component of the promoters of plant genes?

The structural requirements of the ocs-element, a promoter element in several genes transferred to the host plant nucleus by Agrobacterium tumefaciens and certain DNA viruses, have been further characterized both in vitro and in vivo. Two adjacent and functionally identical protein-binding sites separated by an exact number of nucleotides are required for in vivo activity of the ocs-element. Plant pathogens have presumably recruited cellular transcription factors that interact with these binding sites to drive the high-level expression of their essential genes. Our functional analyses of the ocs-elements from two pathogen promoters define the structure of a sequence motif that might also be expected to occur in plant nuclear genes, and a search of the plant gene database has identified a number of plant gene promoters that contain sequences that resemble the ocs-element. These sequences were analysed for their ability both to bind the maize nuclear protein OCSTF and to activate transcription of an inactive promoter. A functional ocs-element was identified in only one of the plant genes, the soybean heat-shock gene, Gmhsp26-A. The apparent rarity of the ocs-element in plant genes contrasts with its frequent use by pathogens that transform the plant nucleus. Sequences resembling half of an ocs-element, on the other hand, are common in plant promoters and may form part of multi-element control motifs with a variety of regulatory functions. Plant pathogens may, therefore, have evolved to circumvent tight regulatory control of their promoters by the host by duplicating the half ocs-element promoter motifs to take advantage of the ubiquitous ocs-element-binding transcription factors in plants.

OCSBF-1, a maize ocs enhancer binding factor: isolation and expression during development.

The ocs-elements comprise a family of related 20-base pair DNA sequences with dyad symmetry that are functional components of the promoters of several genes introduced into the plant nucleus by Agrobacterium transformation or infection by DNA viruses. We describe the isolation and characterization of a maize cDNA that encodes a protein, OCSBF-1, that binds specifically to ocs-element sequences. The 21-kilodalton OCSBF-1 protein was encoded by a single copy, intron-less gene. The gene was differentially expressed in maize plants. Developing leaves had a gradient of OCSBF-1 mRNA with the basal portion of the leaves, which contain dividing and differentiating cells, having 40-fold to 50-fold higher levels of OCSBF-1 transcripts than the apical portion of the leaves, where the cells are fully differentiated. Roots and shoots of young plants had levels of OCSBF-1 mRNA similar to the basal portions of developing leaves. OCSBF-1 contained a small basic amino acid region and a potential leucine zipper motif homologous to the DNA-binding domains of the basic region-leucine zipper family of transcription factors such as Jun and GCN4. A truncated protein with the amino-terminal 76 amino acids of OCSBF-1, encompassing the basic domain and leucine zipper motif, still bound to ocs-element sequences in vitro. OCSBF-1 was able to bind to a site within each half of the ocs-element as well as to animal AP-1 and CREB sites.

A DNA-binding protein factor recognizes two binding domains within the octopine synthase enhancer element.

A protein that binds to the enhancing element of the octopine synthase gene has been identified in nuclear extracts from maize cell suspension cultures. Two protein-DNA complexes are distinguishable by electrophoretic mobility in gel retardation assays. Footprint analyses of these low and high molecular weight complexes show, respectively, half and complete protection of the ocs-element DNA from cleavage by methidiumpropyl-EDTA.FE(II). Two lines of evidence indicate that the element has two recognition sites, each of which can bind identical protein units. Elements that are mutated in one or the other half and form only the low molecular weight complex interfere with the formation of both the low and high molecular weight complexes by the wild-type element. Protein isolated from a complex with only one binding site occupied can bind to the wild-type ocs-element and generate complexes with protein occupying one or both binding sites. Occupation of both sites of the ocs-element is a prerequisite for transcriptional enhancement.

The ocs-element is a component of the promoters of several T-DNA and plant viral genes.

The ocs-element is an enhancer element first identified in the promoter of the octopine synthase gene (OCS) where it occurs as a 16 bp palindromic sequence. The transcriptional enhancing activity of the ocs-element correlated with in vitro binding of a transcription factor. We have now identified ocs-elements in the promoter regions of six other T-DNA genes involved in opine synthesis and three plant viral promoters including the 35S promoter of cauliflower mosaic virus. These elements bind the ocs transcription factor in vitro and enhance transcription in plant cells. Comparison of the sequences of these 10 elements has defined a 20 bp consensus sequence, TGACG(T/C)AAG(C/G)(G/A)(A/C)T(G/T)ACG(T/C)(A/C)(A/C), which includes the 16 bp palindrome in its central region. We propose the name ocs-element for this class of promoter elements of similar sequence and function.

Saturation mutagenesis of the octopine synthase enhancer: correlation of mutant phenotypes with binding of a nuclear protein factor.

A 16-base-pair palindrome from the Agrobacterium tumefaciens octopine synthase gene functions as a constitutive enhancer in plant protoplasts. Degenerate oligonucleotide mutagenesis provided single base substitutions at every position in the element and a number of multiple base substitutions. The effects of these changes were determined in transient expression assays with tobacco and maize protoplasts. The majority of single and double base changes had little effect on the activity of the octopine synthase enhancer, but nearly all mutants with more than two base changes had low to essentially no activity. There were five positions where particular single base changes resulted in a 4- to 10-fold loss in enhancer activity. The distribution of these positions within the palindrome was asymmetric. Single base deletions had essentially no activity, demonstrating that the octopine synthase enhancer cannot tolerate internal changes in spacing. We find a strong correlation between mutant phenotype and reduced binding of a protein factor, suggesting that the DNA-protein complex is responsible for the transcriptional enhancement; the functionally active form of the DNA-protein complex probably involves more than a single protein molecule. The mutants exhibit similar phenotypes in protoplasts of both tobacco and maize, implying conservation of the DNA-protein interactions of the ocs enhancer sequence in monocotyledonous and dicotyledonous plants.

The levels of two distinct species of phytochrome are regulated differently during germination in Avena sativa L.

The abundance and molecular mass of phytochrome in germinating embryos of A. sativa (oat) grown in light or darkness have been monitored using immunoblot and spectrophotometric assays. Immunoblot analysis shows that imbibed but quiescent embryos have two immunochemically distinct species of phytochrome with monomeric molecular masses of 124 and 118 kDa (kdalton). The 118-kDa species has the properties of the 118-kDa phytochrome extracted from fully green oat tissue (J.G. Tokuhisa, S.M. Daniels, P.H. Quail, 1985, Planta 164, 321-332), whereas the 124-kDa polypeptide appears similar to the well-characterized photoreceptor of etiolated tissue. The capacity of antibodies directed against etiolated-oat phytochrome to immunoprecipitate the 124-kDa species but not the 118-kDa species has been exploited to quantitate the levels of each separately over a 72-h time course of germination and seedling development. The abundance of the 124-kDa molecule increases at least 200-fold in etiolated seedlings over 72 h whereas in light-grown seedlings the level of this molecule is relatively constant. In contrast, the amount of the 118-kDa species increases only twofold in both dark- and light-grown seedlings over the same period of time. These data indicate that whereas the abundance of 124-kDa phytochrome is regulated at the protein level by the well-documented, differential stability of the red- and far-red-absorbing forms in vivo, the 118-kDa molecule is present at a low constitutive level, presumably reflecting no such difference in the stability of the two spectral forms.

Phytochrome in green tissue: Spectral and immunochemical evidence for two distinct molecular species of phytochrome in light-grown Avena sativa L.

A method is described for the extraction of phytochrome from chlorophyllous shoots of Avena sativa L. Poly(ethyleneimine) and salt fractionation are used to reduce chlorophyll and to increase the phytochrome concentration sufficiently to permit spectral and immunochemical analyses. The phototransformation difference spectrum of this phytochrome is distinct from that of phytochrome from etiolated shoots in that the maximum in the red region of the difference spectrum is shifted about 15 nm to a shorter wavelength. Immunochemical probing of electroblotted proteins (Western blotting), using a method sensitive to 50 pg, demonstrates the presence of two polypeptides in green tissue that bind antiphytochrome antibodies: a predominant species with a relative molecular mass (Mr) of 118000 and a lesser-abundant 124000-Mr polypeptide. Under nondenaturing conditions all of the 124000-Mr species is immunoprecipitable, but the 118000-Mr species remains in the supernatant. Peptide mapping and immunochemical analysis with monoclonal antibodies show that the 118000-Mr species has structural features that differ from etiolated-oat phytochrome. Mixing experiments show that these structural differences are intrinsic to the molecular species from these two tissues rather than being the result of post-homogenization modifications or interfering substances in the green-tissue extracts. Together the data indicate that the phytochrome that predominates in green-tissue has a polypeptide distinct from the well-characterized molecule from etiolated tissue.