Opposing effects of reduced DNA methylation on flowering time in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Demethylation of DNA promotes flowering in plants from the vernalization-responsive ecotype C24 of Arabidopsis thaliana (L.) Heynh., but delays flowering in the ecotype Landsberg erecta which is not responsive to vernalization. To investigate these contrasting effects of low methylation we have monitored flowering times and expression of two repressors of flowering, FLC and FWA, in low-methylation plants from three late-flowering mutants in the ecotype Landsberg erecta. Demethylation of DNA decreased FLC expression in the vernalization-responsive mutants, but was not associated with a promotion of flowering; rather, in some lines, demethylation delayed flowering. The opposing effects of demethylation could be explained by its differential effect on the expression of two repressors of flowering. FLC was down-regulated in plants with low methylation, promoting flowering, while FWA was activated in response to demethylation, which probably delays the transition to flowering. Expression of the FWA gene did not delay flowering in plants of ecotype C24; our data suggest that the FWA protein of C24 may be non-functional.     

The downregulation of FLOWERING LOCUS C (FLC) expression in plants with low levels of DNA methylation and by vernalization occurs by distinct mechanisms

FLOWERING LOCUS C (FLC), a repressor of flowering, is a major determinant of flowering time in Arabidopsis. FLC expression is repressed by vernalization and in plants with low levels of DNA methylation, resulting in early flowering. This repression is not associated with changes of DNA methylation within the FLC locus in either vernalized plants or plants with low levels of DNA methylation. In both cases, there is a reduction of histone H3 trimethyl-lysine 4 (K4) and acetylation of both histones H3 and H4 around the promoter-translation start of FLC. The expression of the two genes flanking FLC is also repressed in both conditions and repression is associated with decreased histone H3 acetylation. The changes in histone modifications at the FLC gene cluster, which are similar in vernalized plants and in plants with reduced DNA methylation, must arise by different mechanisms. VERNALIZATION 1, VERNALIZATION 2 and VERNALIZATION INSENSITIVE 3 modulate FLC expression in vernalized plants; these proteins play no role in the downregulation of FLC in plants with low levels of DNA methylation. Chimeric FLC::GUS transgenes respond to vernalization but these same transgenes show a position-dependent response to low levels of DNA methylation. In plants with reduced DNA methylation, expression of the five MADS AFFECTING FLOWERING (MAF) genes is repressed, suggesting that DNA methylation alters the expression of a trans-acting regulator common to FLC and members of the related MAF gene family. Our observations suggest that DNA methylation is not part of the vernalization pathway.     

PIE1, an ISWI family gene,is required for FLC activation and floral repression in Arabidopsis

Proper control of the floral transition is critical for reproductive success in flowering plants. In Arabidopsis, FLOWERING LOCUS C (FLC) is a floral repressor upon which multiple floral regulatory pathways converge. Mutations in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) suppress the FLC-mediated delay of flowering as a result of the presence of FRIGIDA or of mutations in autonomous pathway genes. PIE1 is required for high levels of FLC expression in the shoot apex, but it is not required for FLC expression in roots. PIE1 is similar to ATP-dependent, chromatin-remodeling proteins of the ISWI and SWI2/SNF2 family. The role of PIE1 as an activator of FLC is consistent with the general role of ISWI and SWI2/SNF2 family genes as activators of gene expression. The pie1 mutation also causes early flowering in noninductive photoperiods independently of FLC; thus, PIE1 appears to be involved in multiple flowering pathways. PIE1 also plays a role in petal development, as revealed by the suppression of petal defects of the curly leaf mutant by the pie1 mutation.     

A cluster of Arabidopsis genes with a coordinate response to an environmental stimulus

Vernalization, the promotion of flowering after prolonged exposure to low temperatures, is an adaptive response of plants ensuring that flowering occurs at a propitious time in the annual seasonal cycle. In Arabidopsis, FLOWERING LOCUS C (FLC), which encodes a repressor of flowering, is a key gene in the vernalization response; plants with high-FLC expression respond to vernalization by downregulating FLC and thereby flowering at an earlier time. Vernalization has the hallmarks of an epigenetically regulated process. The downregulation of FLC by low temperatures is maintained throughout vegetative development but is reset at each generation. During our study of vernalization, we have found that a small gene cluster, including FLC and its two flanking genes, is coordinately regulated in response to genetic modifiers, to the environmental stimulus of vernalization, and in plants with low levels of DNA methylation. Genes encoded on foreign DNA inserted into the cluster also acquire the low-temperature response. At other chromosomal locations, FLC maintains its response to vernalization and imposes a parallel response on a flanking gene. This suggests that FLC contains sequences that confer changes in gene expression extending beyond FLC itself, perhaps through chromatin modification.     

Transgenic cassava resistance to <Emphasis Type="Italic">African cassava mosaic virus</Emphasis> is enhanced by viral DNA-A bidirectional promoter-derived siRNAs

Expression of double-stranded RNA (dsRNA) homologous to virus sequences can effectively interfere with RNA virus infection in plant cells by triggering RNA silencing. Here we applied this approach against a DNA virus, African cassava mosaic virus (ACMV), in its natural host cassava. Transgenic cassava plants were developed to express small interfering RNAs (siRNA) from a CaMV 35S promoter-controlled, intron-containing dsRNA cognate to the common region-containing bidirectional promoter of ACMV DNA-A. In two of three independent transgenic lines, accelerated plant recovery from ACMV-NOg infection was observed, which correlates with the presence of transgene-derived siRNAs 21–24 nt in length. Overall, cassava mosaic disease symptoms were dramatically attenuated in these two lines and less viral DNA accumulation was detected in their leaves than in those of wild-type plants. In a transient replication assay using leaf disks from the two transgenic lines, strongly reduced accumulation of viral single-stranded DNA was observed. Our study suggests that a natural RNA silencing mechanism targeting DNA viruses through production of virus-derived siRNAs is turned on earlier and more efficiently in transgenic plants expressing dsRNA cognate to the viral promoter and common region.     

The effect of the floral repressor FLC on the timing and progression of vegetative phase change in Arabidopsis

Plants undergo two major post-embryonic developmental transitions--the juvenile-to-adult vegetative transition (vegetative phase change) and the adult-to-reproductive transition (flowering). In woody plants, these transitions can be separated by years, but in herbaceous species they are often very close together, making it difficult to differentiate the effects of vegetative phase change and floral induction on vegetative development. To distinguish between these factors, we have compared the vegetative morphology of plants highly expressing the floral repressor FLC (FRI;FLC) with plants mutant for this gene (FRI;flc-3) under both photoinductive (long day, LD and night interruption, NI) and non-photoinductive (short day, SD) conditions. We show that the onset of abaxial trichome production is insensitive to floral induction, but the distribution and overall number of abaxial trichomes, as well as several other leaf traits associated with vegetative change, are strongly influenced by flowering. Most of the major differences in leaf morphology between FRI;FLC and FRI;flc-3 plants grown in LD can be attributed to the early flowering phenotype of FRI;flc-3, because these differences are not apparent in plants grown in SD. These include differences in leaf size, hydathode number and the distribution of abaxial trichomes along the length of the leaf. Leaf shape and the total number of abaxial trichomes are affected by FLC independently of its effect on flowering. Our results demonstrate that the onset and the progression of vegetative phase change are regulated by different combinations of endogenous and environmental factors, and reveal a role for FLC in vegetative development.     

Vernalization sensitivity in Arabidopsis thaliana (Brassicaceae): the effects of latitude and FLC variation

Latitudinal variation in climate is predicted to select for latitudinal differentiation in sensitivity to the environmental cues that signal plants to flower at the appropriate time for a given climate. In Arabidopsis thaliana, flowering is promoted by exposure to cold temperatures (vernalization), and several vernalization pathway loci are known. To test whether natural variation in vernalization sensitivity could account for a previously observed latitudinal cline in flowering time in A. thaliana, we exposed 21 European accessions to 0, 10, 20, or 30 d of vernalization and observed leaf number at flowering under short days in a growth chamber. We observed a significant latitudinal cline in vernalization sensitivity: southern accessions were more sensitive to vernalization than northern accessions. In addition, accessions that were late flowering in the absence of vernalization were more sensitive to vernalization cues. Allelic variation at the flowering time regulatory gene FLC was not associated with mean vernalization sensitivity, but one allele class exhibited greater variance in vernalization sensitivity.     

FRIGIDA-independent variation in flowering time of natural Arabidopsis thaliana accessions

FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) are two genes that, unless plants are vernalized, greatly delay flowering time in Arabidopsis thaliana. Natural loss-of-function mutations in FRI cause the early flowering growth habits of many A. thaliana accessions. To quantify the variation among wild accessions due to FRI, and to identify additional genetic loci in wild accessions that influence flowering time, we surveyed the flowering times of 145 accessions in long-day photoperiods, with and without a 30-day vernalization treatment, and genotyped them for two common natural lesions in FRI. FRI is disrupted in at least 84 of the accessions, accounting for only approximately 40% of the flowering-time variation in long days. During efforts to dissect the causes for variation that are independent of known dysfunctional FRI alleles, we found new loss-of-function alleles in FLC, as well as late-flowering alleles that do not map to FRI or FLC. An FLC nonsense mutation was found in the early flowering Van-0 accession, which has otherwise functional FRI. In contrast, Lz-0 flowers late because of high levels of FLC expression, even though it has a deletion in FRI. Finally, eXtreme array mapping identified genomic regions linked to the vernalization-independent, late-flowering habit of Bur-0, which has an alternatively spliced FLC allele that behaves as a null allele.     

Leaf Nitrate Reductase, d-Ribulose-1,5-bisphosphate Carboxylase, and Root Nodule Development of Genetic Male-Sterile and Fertile Soybean Isolines

The objectives of this study were to determine the effect of pod and seed development on leaf chlorophyll concentration, and on activities of leaf ribulose bisphosphate carboxylase, leaf nitrate reductase, and root nodule acetylene reduction in field-grown soybean (Glycine max [L.] Merr.). Two genetic male-sterile lines and their fertile counterparts (Williams and Clark 63) were compared in both 1978 and 1979. Two additional lines (Wells × Beeson and Wells × Corsoy) were compared in 1979.

The expression of male-sterile character was nearly complete as very little outcrossing due to insect pollinators was observed. Male-sterile plants showed a delayed late season decline in leaf chlorophyll content and ribulose bisphosphate carboxylase activity when compared with fertile plants. A slight delay in the loss of in vivo leaf nitrate reductase activity was also observed for male-sterile plants. Root nodule fresh weight and acetylene reduction activity declined slightly more rapidly for fertile lines than for male-sterile lines in both years with differences significant on the last two to three sampling dates as leaf loss occurred in the control plants.

Seed development was found to increase slightly, the rate of decline of metabolic activity in fertile lines compared with that of male-sterile lines. However, pod development was not an a priori requirement for leaf and root nodule senescence. Male-sterile plants also lost photosynthetic and nitrogen metabolic competence, but at a slower rate. These results support the concept that pod and seed development does not signal monocarpic senescence per se but rather affects the rate at which senescence occurs after flowering.

     

Determination of the Length of the Vegetative and Pre-floral Stages in the Day-Neutral Plant Helianthus annuus by Chilling Pulses

Helianthus annuus seedlings grown in an 18 h day at 28 ?C wereexposed to one 6 d chilling pulse of 12 ?C, at spaced timesduring the first 21 d from sowing. At 2 d intervals, the terminalbuds of 5 plants were dissected to determine leaf number andto score the vegetative or flowering state of the shoot apex.It was found that, while the rate of leaf initiation was reducedequally by each chilling pulse, pulses commencing on days 9or 12 reduced the total leaf number from 30 to 26, while pulsesapplied earlier had little effect. This variation is interpretedin terms of the time available for leaf production. The apicesof control plants commenced the visible transition to flowering16 d after sowing. Chilling pulses applied from days 3 or 6delayed this transition by about 5 d, whereas later pulses causedonly a 1•5 d delay. In a second experiment, where the chillingwas reduced to 2 d duration, it was again found that chillingdelayed flowering during the first 8 d and was progressivelyless effective when applied later. From this variation in temperaturesensitivity it is proposed that chilling sunflower plants immediatelyafter sowing delays flowering by extending the vegetative phaseof growth and so delaying the attainment of a ‘ripenessto flower’ state that appears to coincide with the expansionof the first pair of leaves. From day 8 onwards processes leadingto flowering that are relatively temperature insensitive apparentlybecome dominant in the apex and result in visible signs of flowering8 d later, although during this transitional stage leaf primordiacontinue to be initiated on the flanks of the apex.     

Efficient Down-Regulation of the Major Vegetative Storage Protein Genes in Transgenic Soybean Does Not Compromise Plant Productivity

Soybean (Glycine max L. Merr.) contains two related and abundant proteins, VSP alpha and VSP beta, that have been called vegetative storage proteins (VSP) based on their pattern of accumulation, degradation, tissue localization, and other characteristics. To determine whether these proteins play a critical role in sequestering N and other nutrients during early plant development, a VspA antisense gene construct was used to create transgenic plants in which VSP expression was suppressed in leaves, flowers, and seed pods. Total VSP was reduced at least 50-fold due to a 100-fold reduction in VSP alpha and a 10-fold reduction in VSP beta. Transgenic lines were grown in replicated yield trials in the field in Nebraska during the summer of 1999 and seed harvested from the lines was analyzed for yield, protein, oil, and amino acid composition. No significant difference (alpha = 0.05) was found between down-regulated lines and controls for any of the traits tested. Young leaves of antisense plants grown in the greenhouse contained around 3% less soluble leaf protein than controls at the time of flowering. However, total leaf N did not vary. Withdrawing N from plants during seed fill did not alter final seed protein content of antisense lines compared with controls. These results indicate that the VSPs play little if any direct role in overall plant productivity under typical growth conditions. The lack of VSPs in antisense plants might be partially compensated for by increases in other proteins and/or non-protein N. The results also suggest that the VSPs could be genetically engineered or replaced without deleterious effects.     

Identification of a MADS-box gene, FLOWERING LOCUS M, that represses flowering

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.     

DNA,RNA, AND PROTEIN COMPARISONS BETWEEN NODULATED AND NON-NODULATED MALE-STERILE AND MALE-FERTILE GENOTYPES OF SOYBEAN (GLYCINE MAX L.)

Four soybean (Glycine max L. Merr.) lines isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁) were developed to study the effects of reproductive development and nitrogen source on the nucleic acid and protein levels within the leaves. Changes in DNA, RNA, protein, and cellular viability were measured from flowering (77 days after emergence) until maturity (147 days after emergence) in leaves of nodulated and non-nodulated male-sterile and fertile soybean genotypes. Leaf nuclei from the sterile genotypes yielded DNA amounts that were significantly higher than those from the fertile lines. The average DNA values for the nodulated sterile and nodulated fertile lines at 147 days after emergence were 7.01 and 2.45 picograms, respectively. The average 2C DNA amount as determined from dividing root-tip nuclei was 2.83 picograms, which indicated occurrence of endopolyploid mechanisms in the sterile lines and age-related loss of DNA in fertile lines. Similar to DNA findings, the RNA and protein values in the sterile lines were significantly higher than those values observed in the fertile lines, suggesting an increased capacity to synthesize protein. The soybean leaf nuclear DNA declined, especially in the fertile lines in terms of the percent endopolyploid nuclei as well as the average DNA content during maturation. The DNA decline in leaves of fertile genotypes suggests that the leaves may be exporting nucleosides and phosphates to the seeds during embryo formation. In the sterile lines, due to the reduced pod-set, these ready reserves of nucleosides and phosphates tended to accumulate in the chromatin of the leaf nucleus as manifested by the DNA specific Feulgen stain. By the end of the study (147 days after emergence), the nodulated fertile genotypes had experienced a dramatic loss in DNA, RNA, and protein. The nodulated sterile genotypes, however, indicated 65% more DNA, 59% more RNA, and 53% more protein as compared to the nodulated fertile genotypes at 147 days after emergence. The sterile lines also indicated the slowest increase in the death of cells, while the fertile lines indicated the fastest increase in nonviable cells, as shown by trypan blue staining. The fertile lines displayed normal monocarpic senescence throughout the study. The reproductive structures of fertile plants utilized the molecules in seed production, whereas in the sterile lines, these accumulated in leaf cells.     

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.