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ABSTRACT The promotion of flowering in response to a prolonged exposure to cold temperatures (i.e. winter) is a useful adaptation for plant species that flower in the spring. This promotion is known as vernalization and results in a permanent memory of cold exposure. While the physiology of vernalization has been extensively studied in many species, the molecular mechanism of vernalization remains largely unknown. Recent studies, however, have revealed some of the molecular events that create the requirement for vernalization. In Arabidopsis, naturally occurring late-flowering ecotypes and plants containing late-flowering mutations in the autonomous floral-promotion pathway are relatively late flowering unless cold treated. The vernalization requirement of these late-flowering ecotypes and autonomous-pathway mutants is largely created by an upregulation of the floral inhibitor FLOWERING LOCUS C (FLC). After cold treatment, as imbibed seeds or young seedlings, FLC transcript levels are downregulated and remain low for the remainder of the plant's life, but return to high levels in the next generation. Plants containing a constitutively expressed 35S:FLC construct remain late flowering after cold treatment, indicating that FLC levels must be downregulated for vernalization to be effective. Thus the epigenetic downregulation of FLC appears to be a major target of the vernalization pathway and provides a molecular marker of the vernalized state. [ABSTRACT FROM AUTHOR]

ABSTRACTWe studied the impact of delayed leaf senescence on the functioning of plants growing under conditions of nitrogen remobilization. Interactions between cytokinin metabolism, Rubisco and protein levels, photosynthesis and plant nitrogen partitioning were studied in transgenic tobacco (Nicotiana tabacum L.) plants showing delayed leaf senescence through a novel type of enhanced cytokinin syn-thesis, i.e. targeted to senescing leaves and negatively auto-regulated (PSAG12–IPT), thus preventing developmental abnormalities. Plants were grown with growth-limiting nitrogen supply. Compared to the wild-type, endogenous levels of free zeatin (Z)- and Z riboside (ZR)-type cytokinins were increased up to 15-fold (total ZR up to 100-fold) in senescing leaves, and twofold in younger leaves of PSAG12–IPT. In these plants, the senescence-associated declines in N, protein and Rubisco levels and photosynthesis rates were delayed. Senescing leaves accumulated more (15N-labelled) N than younger leaves, associated with reduced shoot N accumulation (–60%) and a partially inverted canopy N profile in PSAG12–IPT plants. While root N accumulation was not affected, N translocation to non-senescing leaves was progressively reduced. We discuss potential consequences of these modified sink–source relations, associated with delayed leaf senescence, for plant productivity and the efficiency of utilization of light and minerals. [ABSTRACT FROM AUTHOR]

FLOWERING LOCUS C (FLC) is a major regulator of flowering responses to seasonal environmental factors. Here, we document that FLC also regulates another major life-history transition-seed germination, and that natural variation at the FLC locus and in FLC expression is associated with natural variation in temperature-dependent germination. FLC-mediated germination acts through additional genes in the flowering pathway (FT, SOC1, and AP1) before involving the abscisic acid catabolic pathway (via CYP707A2) and gibberellins biosynthetic pathway (via GA20ox1) in seeds. Also, FLC regulation of germination is largely maternally controlled, with FLC peaking and FT, SOC1, and AP1 levels declining at late stages of seed maturation. High FLC expression during seed maturation is associated with altered expression of hormonal genes (CYP707A2 and GA20ox1) in germinating seeds, indicating that gene expression before the physiological independence of seeds can influence gene expression well after any physical connection between maternal plants and seeds exists. The major role of FLC in temperature-dependent germination documented here reveals a much broader adaptive significance of natural variation in FLC. Therefore, pleiotropy between these major life stages likely influences patterns of natural selection on this important gene, making FLC a promising case for examining how pleiotropy influences adaptive evolution., Organismic and Evolutionary Biology, Version of Record

FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway previously identified. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior.

The MADS domain--containing transcription factor FLOWERING LOCUS C (FLC) acts as an inhibitor of flowering and is a convergence point for several pathways that regulate flowering time in Arabidopsis. In naturally occurring late-flowering ecotypes, the FRIGIDA (FRI) gene acts to increase FLC levels, whereas the autonomous floral promotion pathway and vernalization act to reduce FLC expression. Previous work has shown that the Landsberg erecta allele of FLC, which is not a null allele, is able to partially suppress the late-flowering phenotype of FRIGIDA and mutations in the autonomous pathway. In this study, using a null allele of FLC, we show that the late-flowering phenotype of FRIGIDA and autonomous pathway mutants are eliminated in the absence of FLC activity. In addition, we have found that the downregulation of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 by FRI and autonomous pathway mutants also is mediated by FLC. Complete loss of FLC function, however, does not eliminate the effect of vernalization. Thus, FRI and the autonomous pathway may act solely to regulate FLC expression, whereas vernalization is able to promote flowering via FLC-dependent and FLC-independent mechanisms.

The timing of flowering is important for the reproductive success of plants. Here we describe the identification and characterization of a new MADS-box gene, FLOWERING LOCUS M (FLM), which is involved in the transition from vegetative to reproductive development. FLM is similar in amino-acid sequence to FLC, another MADS-box gene involved in flowering-time control. flm mutants are early flowering in both inductive and non-inductive photoperiods, and flowering time is sensitive to FLM dosage. FLM overexpression produces late-flowering plants. Thus FLM acts as an inhibitor of flowering. FLM is expressed in areas of cell division such as root and shoot apical regions and leaf primordia.

The gibberellin (GA) 20-oxidase encoded by Arabidopsis GA5 catalyzes the synthesis of active GAs. GA5 is a regulatory step in GA biosynthesis as GA5 mRNA levels are negatively regulated by its bioactive GA products. A fusion between the GA5 promoter and the firefly luciferase reporter (GA5-LUC) was shown to be similarly regulated, indicating GA feedback of GA5 occurs at the transcriptional level. The fidelity of the GA5-LUC reporter permitted a fusion genetic screen to identify mutants altered in transgene expression. This bioimaging screen identified two types of recessive mutants with increased LUC activity and apparent GA-related growth phenotypes, a dwarf (lue1) and two late flowering mutants (fpa1-3 and fpa1-4). Mutant progeny exhibited altered levels of LUC and of endogenous GA5 and other GA-regulated mRNAs. SSLP-based mapping localized lue1 to chromosome I near the ga2 locus, although complementation analyzes showed that lue1 is not allelic to ga2. Mapping and complementation analyzes showed that the late flowering mutants are allelic to fpa1. This provides genetic evidence for crosstalk between the autonomous and gibberellin-dependent flowering pathways.

The molecular biology of flowering has been most extensively studied in the quantitative long-day plant Arabidopsis thaliana. The Arabidopsis LUMINIDEPENDENS (LD) gene encodes a potential transcriptional regulator that acts as a positive effector of flowering, at least in part through suppression of the floral inhibitor gene FLC. As an initial step to explore the conservation of the molecular mechanisms of flowering among plants of various flowering habits, and to further investigate the molecular action(s) of LD, we have identified a gene from maize (Zea mays) that is closely related to Arabidopsis LD. The major product of this gene, which we have designated ZmLD for Zea mays LUMINIDEPENDENS, contains four conserved regions that may constitute functionally important components of the LD proteins. One of these regions closely resembles the canonical homeodomain. The ZmLD gene exists as a single copy in the maize genome, and generates a major ca. 4.0 kb transcript, and a minor ca. 2.6 kb transcript that results from alternative transcriptional termination. The 4.0 kb ZmLD alpha transcript accumulated to highest levels in proliferative tissues, including the shoot apex and developing inflorescences. Expression of ZnLD alpha under control of the Arabidopsis LD promoter in transgenic Arabidopsis caused developmental defects similar to those conferred by loss-of-function mutations in a class of genes involved in maintaining the proliferative nature of the shoot, inflorescence, and floral meristems. These effects were not influenced markedly by the activities of the Arabidopsis LD or FLC genes. We consider the implications for the conservation of LD function between maize and Arabidopsis.

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.

We have analysed the circadian rhythm of Arabidopsis thaliana leaf movements in the accession Cvi from the Cape Verde Islands, and in the commonly used laboratory strains Columbia (Col) and Landsberg (erecta) (Ler), which originated in Northern Europe. The parental lines have similar rhythmic periods, but the progeny of crosses among them reveal extensive variation for this trait. An analysis of 48 Ler/Cvi recombinant inbred lines (RILs) and a further 30 Ler/Col RILs allowed us to locate four putative quantitative trait loci (QTLs) that control the period of the circadian clock. Near-isogenic lines (NILs) that contain a QTL in a small, defined chromo- somal region allowed us to confirm the phenotypic effect and to map the positions of three period QTLs, designated ESPRESSO, NON TROPPO and RALENTANDO. Quantitative trait loci at the locations of RALENTANDO and of a fourth QTL, ANDANTE, were identified in both Ler/Cvi and Ler/Col RIL populations. Some QTLs for circadian period are closely linked to loci that control flowering time, including FLC. We show that flc mutations shorten the circadian period such that the known allelic variation in the MADS-box gene FLC can account for the ANDANTE QTL. The QTLs ESPRESSO and RALENTANDO identify new genes that regulate the Arabidopsis circadian system in nature, one of which may be the flowering-time gene GIGANTEA.

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., (Copyright 1999 Wiley-Liss, Inc.)

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.

SAG12 is a developmentally controlled, senescence-specific gene from Arabidopsis which encodes a cysteine protease. Using SAG12 as a probe, we isolated two SAG12 homologues (BnSAG12-1 and BnSAG12-2) from Brassica napus. Structural comparisons and expression studies indicate that these two genes are orthologues of SAG12. The expression patterns of BnSAG12-1 and BnSAG12-2 in Arabidopsis demonstrate that the senescence-specific regulation of this class of cysteine proteases is conserved across these species. Gel-shift assays using the essential promoter regions of SAG12, BnSAG12-1, and BnSAG12-2 show that the extent of binding of a senescence-specific, DNA-binding protein from Arabidopsis is proportional to the expression levels of these genes in Arabidopsis. Therefore, the expression levels of these genes may reflect the affinities of the senescence-specific DNA-binding protein for the promoter element.

To determine the range of gene activities associated with leaf senescence, we have identified genes that show preferential transcript accumulation during this developmental stage. The mRNA levels of a diverse array of gene products increases during leaf senescence, including a protease, a ribosomal protein, two cinnamyl alcohol dehydrogenases, a nitrilase and glyoxalase II. Two of the genes identified are known to be pathogen-induced. The senescence specificity of each gene was determined by characterization of transcript accumulation during leaf development and in different tissues. The increased expression of nitrilase in senescent leaves is paralleled by an increase in free indole-3-acetic acid (IAA) levels. Additionally, we have demonstrated that the induction of defense-related genes during leaf senescence is pathogen-independent and that salicylic acid accumulation is not essential for this induction. Our data indicate that the induction of certain genes involved in plant defense responses is a component of the leaf senescence program.

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.

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.

Mutations in the LUMINIDEPENDENS (LD) gene of Arabidopsis thaliana (L.) Heynh. (Arabidopsis) confer a late-flowering phenotype, indicating that LD normally functions to promote the floral transition. RNA and protein blot analyses, along with the analysis of transgenic plants containing a fusion between a genomic fragment of LD and the reporter gene uidA (GUS), indicate that LD is expressed primarily ipical proliferative regions of the shoot and root, including the shoot apical meristem and leaf primordia. Subcellular localization studies indicate that LD is a nuclear protein, consistent with its previously proposed transcriptional regulatory role. We have also found that in an apetala1 cauliflower (ap1 cal) background the ld mutation converts the reproductive shoot apex to a more vegetative state, a phenotype that is similar to that seen for the leafy (lfy) mutant. Furthermore, in situ hybridization analysis indicates that LFY levels are drastically reduced at the apex of ld ap1 cal plants after bolting. These data are consistent with the idea that at least one function of LD is to participate in the regulation of LFY.

The expression of several Arabidopsis thaliana senescence-associated genes (SAGs) in attached and/or detached leaves was compared in response to age, dehydration, darkness, abscisic acid, cytokinin, and ethylene treatments. Most of the SAGs responded to most of the treatments in a similar fashion. Detachment in darkness and ethylene were the strongest inducers of both SAGs and visible yellowing. Detachment in light was also a strong inducer of SAGs, but not of visible yellowing. The other treatments varied more in their effects on individual SAGs. Responses were examined in both older and younger leaves, and generally were much stronger in the older ones. Individual SAGs differed from the norms in different ways, however, suggesting that their gene products play a role in overlapping but not identical circumstances. Some SAGs responded quickly to treatments, which may indicate a direct response. Others responded more slowly, which may indicate an indirect response via treatment-induced senescence. Four new SAGs were isolated as part of this work, one of which shows strong similarity to late embryogenesis-abundant (Lea) genes.

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.

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.

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.