Zebularine treatment is associated with deletion of FT‐B1 leading to an increase in spikelet number in bread wheat

The number of rachis nodes (spikelets) on a wheat spike is a component of grain yield that correlates with flowering time. The genetic basis regulating flowering in cereals is well understood, but there are reports that flowering time can be modified at a high frequency by selective breeding, suggesting that it may be regulated by both epigenetic and genetic mechanisms. We investigated the role of DNA methylation in regulating spikelet number and flowering time by treating a semi‐spring wheat with the demethylating agent, Zebularine. Three lines with a heritable increase in spikelet number were identified. The molecular basis for increased spikelet number was not determined in 2 lines, but the phenotype showed non‐Mendelian inheritance, suggesting that it could have an epigenetic basis. In the remaining line, the increased spikelet phenotype behaved as a Mendelian recessive trait and late flowering was associated with a deletion encompassing the floral promoter, FT‐B1. Deletion of FT‐B1 delayed the transition to reproductive growth, extended the duration of spike development, and increased spikelet number under different temperature regimes and photoperiod. Transiently disrupting DNA methylation can generate novel flowering behaviour in wheat, but these changes may not be sufficiently stable for use in breeding programs.     

The Arabidopsis TALE homeobox gene ATH1 controls floral competency through positive regulation of FLC

Floral induction is controlled by a plethora of genes acting in different pathways that either repress or promote floral transition at the shoot apical meristem (SAM). During vegetative development high levels of floral repressors maintain the Arabidopsis SAM as incompetent to respond to promoting factors. Among these repressors, FLOWERING LOCUS C (FLC) is the most prominent. The processes underlying downregulation of FLC in response to environmental and developmental signals have been elucidated in considerable detail. However, the basal induction of FLC and its upregulation by FRIGIDA (FRI) are still poorly understood. Here we report the functional characterization of the ARABIDOPSIS THALIANA HOMEOBOX 1 (ATH1) gene. A function of ATH1 in floral repression is suggested by a gradual downregulation of ATH1 in the SAM prior to floral transition. Further evidence for such a function of ATH1 is provided by the vernalization-sensitive late flowering of plants that constitutively express ATH1. Analysis of lines that differ in FRI and/or FLC allele strength show that this late flowering is caused by upregulation of FLC as a result of synergism between ATH1 overexpression and FRI. Lack of ATH1, however, results in attenuated FLC levels independently of FRI, suggesting that ATH1 acts as a general activator of FLC expression. This is further corroborated by a reduction of FLC-mediated late flowering in fca-1 and fve-1 autonomous pathway backgrounds when combined with ath1. Since other floral repressors of the FLC clade are not significantly affected by ATH1, we conclude that ATH1 controls floral competency as a specific activator of FLC expression.     

Developmental responses of bread wheat to changes in ambient temperature following deletion of a locus that includes FLOWERING LOCUS T1

FLOWERING LOCUS T (FT) is a central integrator of environmental signals that regulates the timing of vegetative to reproductive transition in flowering plants. In model plants, these environmental signals have been shown to include photoperiod, vernalization, and ambient temperature pathways, and in crop species, the integration of the ambient temperature pathway remains less well understood. In hexaploid wheat, at least 5 FT‐like genes have been identified, each with a copy on the A, B, and D genomes. Here, we report the characterization of FT‐B1 through analysis of FT‐B1 null and overexpression genotypes under different ambient temperature conditions. This analysis has identified that the FT‐B1 alleles perform differently under diverse environmental conditions; most notably, the FT‐B1 null produces an increase in spikelet and tiller number when grown at lower temperature conditions. Additionally, absence of FT‐B1 facilitates more rapid germination under both light and dark conditions. These results provide an opportunity to understand the FT‐dependent pathways that underpin key responses of wheat development to changes in ambient temperature. This is particularly important for wheat, for which development and grain productivity are sensitive to changes in temperature.     

Interaction of gibberellic acid and 24-epibrassinolide in the regulation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> seedling scotomorphogenesis

The effects of GA₃, 24-epibrassinolide (EBL), and their combination on morphogenesis of Arabidopsis thaliana (L.) Heynh seven-day-old seedlings were studied. Four plant lines were analyzed: wild type Ler and ga4-1 mutant, belonging to the Landsberg erecta ecotype and wild type Col and det2 mutant, both of the Columbia ecotype. In ga4-1 and det2, GA_4/1-and brassinosteroid-deficient mutants, the highest hypocotyl growth response to the lack of hormones was noted. The cotyledon shape and size were dependent on EBL, and the root length was both GA₃-and EBL-regulated, indicating organ specificities in the responses to these hormones. Simultaneous treatment of dark-grown plants with GA₃ and EBL exerted an additive stimulatory effect on the root growth of det2, reduced the inhibitory effect of EBL on hypocotyl elongation of ga4-1, and enhanced the effect of EBL on hypocotyl and cotyledon elongation of det2.     

Quantitative effects of vernalization on FLC and SOC1 expression

Prolonged exposure to cold results in early flowering in Arabidopsis winter annual ecotypes, with longer exposures resulting in a greater promotion of flowering than shorter exposures. The promotion of flowering is mediated through an epigenetic down-regulation of the floral repressor FLOWERING LOCUS C (FLC). We present results that provide an insight into the quantitative regulation of FLC by vernalization. Analysis of the effect of seed or plant cold treatment on FLC expression indicates that the time-dependent nature of vernalization on FLC expression is mediated through the extent of the initial repression of FLC and not by affecting the ability to maintain the repressed state. In the over-expression mutant flc-11, the time-dependent repression of FLC correlates with the proportional deacetylation of histone H3. Our results indicate that sequences within intron 1 and the activities of both VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2) are required for efficient establishment of FLC repression; however, VRN1 and VRN2 are not required for maintenance of the repressed state during growth after the cold exposure. SUPPRESSOR OF OVER-EXPRESSION OF CO 1 (SOC1), a downstream target of FLC, is quantitatively induced by vernalization in a reciprocal manner to FLC. In addition, we show that SOC1 undergoes an acute induction by both short and long cold exposures.     

Overexpression of a Kunitz-type trypsin inhibitor (AtKTI1) causes early flowering in Arabidopsis

An early flowering mutant of Arabidopsis, elf32-D was isolated from activation tagging screening. The mutant flowered earlier than wild type under both long day and short day conditions. The mutant phenotype was caused by overexpression of a Kunitz-type trypsin inhibitor gene (AtKTI1). The expression of AtKTI1 was detected in leaves, flowers, siliques and roots. In the vegetative state, no change of flowering integrator gene expression was observed for AtKTI1 overexpressing plants. In contrast, at the reproductive stage, its overexpression resulted in the down-regulation of FLC, a strong floral repressor which integrates the autonomous and vernalization pathways and also the up-regulation of FT and AP1, which are downstream floral integrator genes. It is probable that the AtKTI1 overexpression inhibits components of the flowering signaling pathway upstream of FLC, eventually regulating expression of FLC, or causing perturbations in plant metabolism and thus indirectly affecting flowering.     

Polycomb proteins regulate the quantitative induction of VERNALIZATION INSENSITIVE 3 in response to low temperatures

Vernalization, the promotion of flowering in response to low temperatures, is one of the best characterized examples of epigenetic regulation in plants. The promotion of flowering is proportional to the duration of the cold period, but the mechanism by which plants measure time at low temperatures has been a long‐standing mystery. We show that the quantitative induction of the first gene in the Arabidopsis vernalization pathway, VERNALIZATION INSENSITIVE 3 (VIN3), is regulated by the components of Polycomb Response Complex 2, which trimethylates histone H3 lysine 27 (H3K27me3). In differentiated animal cells, H3K27me3 is mostly associated with long‐term gene repression, whereas, in pluripotent embyonic stem cells, many cell lineage‐specific genes are inactive but exist in bivalent chromatin that carries both active (H3K4me3) and repressive (H3K27me3) marks on the same molecule. During differentiation, bivalent domains are generally resolved to an active or silent state. We found that H3K27me3 maintains VIN3 in a repressed state prior to cold exposure; this mark is not removed during VIN3 induction. Instead, active VIN3 is associated with bivalently marked chromatin. The continued presence of H3K27me3 ensures that induction of VIN3 is proportional to the duration of the cold, and that plants require prolonged cold to promote the transition to flowering. The observation that Polycomb proteins control VIN3 activity defines a new role for Polycomb proteins in regulating the rate of gene induction.     

大豆开花基因GmCO和GmFT的克隆及表达

为了研究大豆光周期反应是否受开花基因CO(CONSTANS)和FT(FLOWERING LOCUS T)调控,采用同源序列法从大豆中分离了CO和FT的同源物GmCO和GmFT.GmCO和GmFT分别编码151和109个氨基酸,与水稻和拟南芥中相关蛋白的氨基酸序列同源性达到70%以上.通过RT-PCR分析GmCO和GmFT在短日照(short daylength,SD)、自然光照(natural light,NL)和长日照(long daylength,LD)处理大豆不同发育阶段叶片中的表达发现,GmCO在LD处理大豆早期发育的叶片中高丰度表达,GmFT在SD和NL处理大豆开花时期的叶片中高丰度表达.上述结果表明,GmCO和GmFT的表达与大豆开花时间及光照长度密切相关,且GmCO抑制GmFT的表达.     

Resetting and regulation of FLOWERING LOCUS C expression during Arabidopsis reproductive development

The epigenetic regulation of the floral repressor FLOWERING LOCUS C ( FLC ) is one of the critical factors that determine flowering time in Arabidopsis thaliana . Although many FLC regulators, and their effects on FLC chromatin, have been extensively studied, the epigenetic resetting of FLC has not yet been thoroughly characterized. Here, we investigate the FLC expression during gametogenesis and embryogenesis using FLC::GUS transgenic plants and RNA analysis. Regardless of the epigenetic state in adult plants, FLC expression disappeared in gametophytes. Subsequently, FLC expression was reactivated after fertilization in embryos, but not in the endosperm. Both parental alleles contributed equally to the expression of FLC in embryos. Surprisingly, the reactivation of FLC in early embryos was independent of FRIGIDA (FRI) and SUPPRESSOR OF FRIGIDA 4 (SUF4) activities. Instead, FRI , SUF4 and autonomous-pathway genes determined the level of FLC expression only in late embryogenesis. Many FLC regulators exhibited expression patterns similar to that of FLC , indicating potential roles in FLC reprogramming. An FVE mutation caused ectopic expression of FLC in the endosperm. A mutation in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 caused defects in FLC reactivation in early embryogenesis, and maintenance of full FLC expression in late embryogenesis. We also show that the polycomb group complex components, Fertilization-Independent endosperm and MEDEA, which mediate epigenetic regulation in seeds, are not relevant for FLC reprogramming. Based on our results, we propose that FLC reprogramming is composed of three phases: (i) repression in gametogenesis, (ii) reactivation in early embryogenesis and (iii) maintenance in late embryogenesis.     

RFLP Analysis of the <Emphasis Type="Italic">FLOWERING LOCUS C</Emphasis> Gene in Six <Emphasis Type="Italic">Brassica</Emphasis> Species

The FLOWERING LOCUS C (FLC) gene controls the transition of arabidopsis plants to flowering following cold induction (vernalization). Time to flowering in annual and biennial species of Brassicaceae supposedly depends on the number of FLC copies. We analyzed DNA restriction fragment length polymorphism in six Brassica species with diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes using for a hybridization probe an FLC homolog previously cloned in our laboratory from B. juncea. The characteristic variations in the patterns of restriction fragments corresponded to the genomic composition of Brassica species and, in some cases, correlated with the timing of floral transition.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 399–405.Original Russian Text Copyright © 2005 by Martynov, Khavkin.     

Overexpression of COL9, a CONSTANS-LIKE gene, delays flowering by reducing expression of CO and FT in Arabidopsis thaliana

CONSTANS (CO) is an important floral regulator in the photoperiod pathway, integrating the circadian clock and light signal into a control for flowering time. It is known that CO promotes flowering in Arabidopsis under long-day conditions. CONSTANS-LIKE 9 (COL9) is a member of the CONSTANS-LIKE gene family, encoding a nuclear protein. The expression of COL9 is regulated by the circadian clock in the photoperiod pathway and is detected in various organs. Unexpectedly, overexpression of COL9 in transgenic Arabidopsis resulted in delayed flowering, while co-suppression lines and a transferred DNA (T-DNA) knockout line showed earlier flowering under long-day conditions. Overexpression of COL9 did not enhance the late-flowering phenotype in a co mutant background. Double overexpressors produced by overexpression of CO in COL9 transgenic lines showed an early flowering phenotype similar to single CO overexpressors. The pattern of oscillation of a number of circadian-associated genes remained unchanged in the COL9 transgenic lines. Compared with wild-type plants, the abundance of CO and FLOWERING LOCUS T (FT) mRNA was reduced in the COL9 overexpression lines. Our results indicate that COL9 is involved in regulation of flowering time by repressing the expression of CO, concomitantly reducing the expression of FT and delaying floral transition.     

Metabolic transformations of some ent-kaurenes in Gibberella fujikuroi

The conversion of ent-kaur-16-enes to gibberellic acid in Gibberella fujikuroi is blocked by A-ring modifications. Thus ent-3β-hydroxykaur-16-en-19-yl succinate gives good conversion (46%) to the 7β-hydroxy derivative.* Under the same conditions the 3β-epimer gives 7β- or 6α-hydroxylation and the former occurs for the 3-oxo analogue. The succinoyloxy function acts as a less efficient block and ent-kaur-16-en-19-yl succinate is converted to 7β-hydroxy and 6β,7β-dihydroxy derivatives along with gibberellic acid. Hydrolysis of the succinate block of the metabolities provides the 7β, 19-diol and 6β,7β, 19-triol. Of this pair only the former was effectively metabolized to gibberellic acid in G. fujikuroi.     

Metabolic engineering of β‐carotene in orange fruit increases its in vivo antioxidant properties

Orange is a major crop and an important source of health‐promoting bioactive compounds. Increasing the levels of specific antioxidants in orange fruit through metabolic engineering could strengthen the fruit's health benefits. In this work, we have afforded enhancing the β‐carotene content of orange fruit through blocking by RNA interference the expression of an endogenous β‐carotene hydroxylase gene (Csβ‐CHX) that is involved in the conversion of β‐carotene into xanthophylls. Additionally, we have simultaneously overexpressed a key regulator gene of flowering transition, the FLOWERING LOCUS T from sweet orange (CsFT), in the transgenic juvenile plants, which allowed us to obtain fruit in an extremely short period of time. Silencing the Csβ‐CHX gene resulted in oranges with a deep yellow (‘golden’) phenotype and significant increases (up to 36‐fold) in β‐carotene content in the pulp. The capacity of β‐carotene‐enriched oranges for protection against oxidative stress in vivo was assessed using Caenorhabditis elegans as experimental animal model. Golden oranges induced a 20% higher antioxidant effect than the isogenic control. This is the first example of the successful metabolic engineering of the β‐carotene content (or the content of any other phytonutrient) in oranges and demonstrates the potential of genetic engineering for the nutritional enhancement of fruit tree crops.     

植物同源结构域指蛋白在拟南芥等十字花科植物春化作用途径中的功能

春化低温处理可以使拟南芥等十字花科植物提前开花,该过程中涉及到一个重要的植物同源结构域指(PHD-finger)蛋白VERNALIZATION INSENSITIVE3(VIN3)。PHD-finger结构域是真核生物中一种进化保守的锌指结构域,通常参与蛋白质之间的相互作用,特别是对核小体组蛋白进行甲基化、乙酰化、磷酸化等修饰。在春化处理过程中,VIN3及其同源基因编码的蛋白都具有PHD-finger结构域,该结构域通过对开花抑制基因FLOWERING LOCUS C染色质组蛋白进行H3K9、H3K27甲基化、H3K9和H3K14去乙酰化等修饰,调节FLC染色质结构状态,使其从松弛状态转变为高度凝缩状态而关闭其功能,从而影响FLC转录活性进而促进开花。以下综述了拟南芥等十字花科植物春化作用途径中PHD-finger蛋白的功能,并且概述了春化作用机制。