Inhibition of leaf senescence by autoregulated production of cytokinin.

Controlling expression of IPT, a gene encoding isopentenyltransferase (the enzyme that catalyzes the rate-limiting step in cytokinin biosynthesis), with a senescence-specific promoter results in the suppression of leaf senescence. Transgenic tobacco plants expressing this chimeric gene do not exhibit the developmental abnormalities usually associated with IPT expression because the system is autoregulatory. Because sufficient cytokinin is produced to retard senescence, the activity of the senescence-specific promoter is attenulated. Senescence-retarded leaves exhibit a prolonged, photosythetically active life-span. This result demonstrates that endogenously produced cytokinin can regulate senescence and provides a system to specifically manipulate the senescence program.

Control of flowering time in plants.

The mechanisms by which the transition to flowering is regulated in plants have been the subject of intense physiological study for many years. Recent studies, particularly in Arabidopsis thaliana, have revealed the genetic complexity of flowering. Flowering appears to be controlled by multiple pathways that are influenced by the environment in which the plant is grown as well as the developmental state of the plant. Several genes that regulate flowering time have been molecularly identified and the effects of altered expression of these genes have contributed greatly to understanding their role in flowering.

Extensive changes in DNA methylation patterns accompany activation of a silent T-DNA ipt gene in Agrobacterium tumefaciens-transformed plant cells.

We crossed a male-sterile, Agrobacterium-transformed Nicotiana tabacum plant that contains a silent, hypermethylated T-DNA ipt oncogene with a normal tobacco plant and found that the methylated state of the ipt gene was stably inherited through meiosis in the offspring. However, when tissues of these plants were placed in cell culture, the ipt gene was spontaneously reactivated in a very small fraction of the cells; if 5-azacytidine was added to the culture medium, ipt gene reactivation occurred at high frequency. We analyzed the pattern of DNA methylation in a region spanning the ipt gene in a line that does not express the ipt gene, in five derivatives of this line that reexpressed the ipt gene either spontaneously or after 5-azacytidine treatment, and in tissues of a sibling of this line that reexpressed ipt spontaneously. We found that the ipt locus was highly methylated in the unexpressed state but that spontaneous or 5-azacytidine-induced reexpression always resulted in extensive demethylation of a region including 5' upstream, coding, and 3' downstream regions of the ipt gene. The role of DNA methylation in gene regulation in this system is discussed.

Delivering copper within plant cells.

Two genes recently identified in Arabidopsis thaliana may be involved in sequestering free copper ions in the cytoplasm and delivering copper to post-Golgi vesicles. The genes COPPER CHAPERONE and RESPONSIVE TO ANTAGONIST1 are homologous to copper-trafficking genes from yeast and humans. This plant copper-delivery pathway is required to create functional ethylene receptors. The pathway may also facilitate the transport of copper from senescing leaf tissue. In addition, several other genes have been identified recently that may have a role in copper salvage during senescence.

Molecular aspects of leaf senescence.

Senescence is the last stage of leaf development and one type of programmed cell death that occurs in plants. The relationships among senescence programs that are induced by a variety of factors have been addressed at a molecular level in recent studies. Furthermore, an overlap between the pathogen-response and senescence programs is beginning to be characterized. The complexity of the senescence program is also evident in studies of senescence-specific gene regulation and the role of photosynthesis and plant hormones in senescence regulation. New molecular-genetic approaches are expected to be useful in unraveling the molecular mechanisms of the leaf senescence program.

Effect of Vernalization, Photoperiod, and Light Quality on the Flowering Phenotype of Arabidopsis Plants Containing the FRIGIDA Gene.

We have compared the flowering response to vernalization, photoperiod, and far-red (FR) light of the Columbia (Col) and Landsberg erecta (Ler) ecotypes of Arabidopsis into which the flowering-time locus FRIGIDA (FRI) has been introgressed with that of the wild types Col, Ler, and San Feliu-2 (Sf-2). In the early-flowering parental ecotypes, Col and Ler, a large decrease in flowering time in response to vernalization was observed only under short-day conditions. However, Sf-2 and the Ler and Col genotypes containing FRI showed a strong response to vernalization when grown in either long days or short days. Although vernalization reduced the responsiveness to photoperiod, plants vernalized for more than 80 d still showed a slight photoperiod response. The effect of FRI on flowering was eliminated by 30 to 40 d of vernalization; subsequently, the response to vernalization in both long days and short days was the same in Col and Ler with or without FRI. FR-light enrichment accelerated flowering in all ecotypes and introgressed lines. However, the FR-light effect was most conspicuous in the FRI-containing plants. Saturation of the vernalization effect eliminated the effect of FR light on flowering, although vernalization did not eliminate the increase of petiole length in FR light.

Ecotype-Specific Expression of a Flowering Mutant Phenotype in Arabidopsis thaliana.

The majority of mutations that delay flowering in Arabidopsis thaliana have been identified in studies of the Landsberg erecta (Ler) ecotype. In this report we describe a gene (referred to as FLD) that, when mutated, delays flowering in the Columbia ecotype but has a minimal phenotype in the Ler genetic background. The late-flowering phenotype of fld mutants requires a non-Ler allele of another gene involved in the control of flowering time, Flowering Locus C. fld mutants retain a photoperiod response, and the flowering time of fld mutants can be reduced by cold treatment and low red/far-red light ratios.

The molecular basis of vernalization in different plant groups.

Timing of flowering is key to the reproductive success of many plants. In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization, the process by which a prolonged exposure to the cold of winter results in competence to flower during the following spring, is an example of the influence of temperature on the timing of flowering. In different groups of plants, there are distinct genes involved in vernalization, indicating that vernalization systems evolved independently in different plant groups. The convergent evolution of vernalization systems is not surprising given that angiosperm families had begun to diverge in warmer paleoclimates in which a vernalization response was not advantageous. Here, we review what is known of the vernalization response in three different plant groups: crucifers (Arabidopsis), Amaranthaceae (sugar beet), and Pooideae (wheat, barley, and Brachypodium distachyon). We also discuss the advantages of using Brachypodium as a model system to study flowering and vernalization in the Pooids. Finally, we discuss the evolution and function of the Ghd7/VRN2 gene family in grasses.

Acceleration of nucleic acid hybridization rate by polyethylene glycol.

The addition of polyethylene glycol to filter-bound nucleic acid hybridization greatly increases the hybridization rate. With single-stranded probes, the increase obtained with polyethylene glycol is significantly greater than that obtained with dextran sulfate. Additionally, polyethylene glycol is easier to manipulate and less expensive than dextran sulfate.

Interaction of FLC and late-flowering mutations in Arabidopsis thaliana.

The phenotype caused by mutations that affect the timing of flowering in Arabidopsis thaliana has been most extensively analyzed in the Landsberg erecta (Ler) genetic background. In Ler, the late-flowering phenotype of FRIGIDA and mutations in LUMINIDEPENDENS is suppressed by the Ler allele of FLC. In this study, the interactions of nine mutations conferring late flowering with the FLC allele of the Columbia ecotype (FLC-Col), which does not suppress late flowering, were examined. The effect on flowering time of combining six of the mutations with FLC-Col was additive; plants containing FLC-Col with fd, gi, fwa, fha, ft, and fe flowered slightly later than plants containing these mutations with the Ler allele of FLC. In contrast, a synergistic effect was observed between FLC-Col and three mutations; fca, fpa, and fve plants became extremely late flowering when combined with FLC-Col. Maximum delay in flowering for the majority of the mutant strains required FLC-Col in a homozygous state, although for fpa and fe a single copy of FLC-Col allowed maximum lateness. In addition, the fd and fe mutations became more dominant in the presence of FLC-Col.

Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation.

We report the isolation of five cDNA clones whose corresponding mRNAs accumulate in cultured soybean cells (Glycine max cv Mandarin) during cytokinin or auxin starvation. The levels of three of these mRNAs decrease rapidly after addition of 5 micromolar zeatin to cytokinin-starved cells or after addition of 10 micromolar alpha-naphthaleneacetic acid to auxin-starved cells. These mRNAs also exhibit various patterns of accumulation in the tissues of intact soybean plants. Partial nucleotide sequence analysis demonstrates that one of the cDNAs in the collection, called SAM46, is 46% identical at the amino acid level to the iron superoxide dismutase gene of Escherichia coli. Expression of this cDNA in Escherichia coli cells results in detectable iron superoxide dismutase activity, confirming the identity of the cDNA.

Expression of an Agrobacterium Ti plasmid gene involved in cytokinin biosynthesis is regulated by virulence loci and induced by plant phenolic compounds.

The nopaline-type Ti plasmid T37 of Agrobacterium tumefaciens carries two distinct genes that encode enzymes involved in cytokinin biosynthesis. In this report, we show that the level of expression of one of these genes was increased dramatically by culture conditions that increased the expression of Ti plasmid virulence genes, including coculture with plant cells or treatment with acetosyringone, a plant phenolic compound. When this nopaline-type Ti plasmid gene was introduced into Agrobacterium strains containing an octopine-type Ti plasmid, similar induction of expression by culture conditions was observed, and analysis of virulence region mutants demonstrated that this induction was under the control of the virA and virG regulatory loci. We further show that induction was strongly pH dependent in octopine strains but, under the conditions examined, pH independent in nopaline strains.

Hormonal control of tobacco crown gall tumor morphology.

The endogenous levels of auxin and cytokinin in teratoma and unorganized tobacco (Nicotiana tabacum L. var Wisconsin #38) crown gall tumor tissues were determined. Teratoma tissues contain levels of auxin and cytokinin favorable for shoot formation, whereas unorganized tumors contain levels of auxin that suppress shoot formation. This conclusion is based upon the observation that when levels of auxin and cytokinin similar to those found in a teratoma were added to the growth medium of nontumorous tobacco tissue, shoot formation resulted; when levels similar to those found in unorganized tumors were added, the normal tissue grew as unorganized callus.

The gibberellic acid biosynthesis mutant ga1-3 of Arabidopsis thaliana is responsive to vernalization.

The Arabidopsis mutant ga1-3 contains a deletion in an enzyme that catalyzes an early step in the synthesis of gibberellic acid. It has been shown that ga1-3 mutant plants cannot flower under 8-h short-day (SD) conditions, even after vernalization. In this article, we present data demonstrating that the ga1-3 mutation does not block the response to vernalization in intermediate photoperiods or in long-day conditions in a late-flowering, vernalization-responsive background. Thus, GA may not have a direct role in the vernalization response in Arabidopsis, but it may be required for an alternate pathway that promotes flowering in noninductive photoperiods.

FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering.

Winter-annual ecotypes of Arabidopsis are relatively late flowering, unless the flowering of these ecotypes is promoted by exposure to cold (vernalization). This vernalization-suppressible, late-flowering phenotype results from the presence of dominant, late-flowering alleles at two loci, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). In this study, we report that flc null mutations result in early flowering, demonstrating that the role of active FLC alleles is to repress flowering. FLC was isolated by positional cloning and found to encode a novel MADS domain protein. The levels of FLC mRNA are regulated positively by FRI and negatively by LUMINIDEPENDENS. FLC is also negatively regulated by vernalization. Overexpression of FLC from a heterologous promoter is sufficient to delay flowering in the absence of an active FRI allele. We propose that the level of FLC activity acts through a rheostat-like mechanism to control flowering time in Arabidopsis and that modulation of FLC expression is a component of the vernalization response.

A robust method for detecting single-nucleotide changes as polymorphic markers by PCR.

Numerous techniques in plant molecular genetic analysis, such as mapping and positional cloning techniques, rely on the availability of molecular markers that can differentiate between alleles at a particular locus. PCR-based cleaved amplified polymorphic sequences (CAPS) markers have been widely used as a means of rapidly and reliably detecting a single-base change that creates a unique restriction site in one of a pair of alleles. However, the majority of single-nucleotide changes do not create such sites and thus cannot be used to create CAPS markers. In this paper, a modification of the CAPS technique that allows detection of most single-nucleotide changes by utilizing mismatched PCR primers is described. The mismatches in the PCR primers, in combination with the single-nucleotide change, create a unique restriction site in one of the alleles.

Identification of a promoter region responsible for the senescence-specific expression of SAG12.

SAG12, an Arabidopsis gene encoding a cysteine protease, is expressed only in senescent tissues. Studies of the expression patterns of a variety of genes showing senescence-specific or senescence-preferential expression indicate that plant senescence involves multiple regulatory pathways. In this study it is shown that the expression of SAG12 is specifically activated by developmentally controlled senescence pathways but not by stress- or hormone-controlled pathways. Using SAG12 as a molecular marker for the study of developmental senescence, we show that cytokinin, auxin, and sugars can repress developmental senescence at the molecular level. Studies using promoter deletions and recombination of promoter fragments indicate that a highly conserved region of the SAG12 promoter is responsible for senescence-specific regulation, while at least two other regions of the SAG12 promoter are important for full promoter activity. Extracts from young and senescent Arabidopsis leaves contain factors that exhibit differential binding to the senescence-responsive promoter element.