Methyl-CpG-binding domain (MBD) proteins in plants

Cytosine methylation is the most prevalent epigenetic modification of plant nuclear DNA, which occurs in symmetrical CpG or CpNpG as well as in non-symmetrical contexts. Intensive studies demonstrated the central role played by cytosine methylation in genome organization, gene expression and in plant growth and development. However, the way by which the methyl group is interpreted into a functional state has only recently begun to be explored with the isolation and characterization of methylated DNA binding proteins capable of binding 5-methylcytosine. These proteins belong to an evolutionary conserved protein family initially described in animals termed methyl-CpG-binding domain (MBD) proteins. Here, we highlight recent advances and present new prospects concerning plant MBD proteins and their possible role in controlling chromatin structure mediated by CpG methylation.     

Alternate Bearing in Citrus: Changes in the Expression of Flowering Control Genes and in Global Gene Expression in ON- versus OFF-Crop Trees

Alternate bearing (AB) is the process in fruit trees by which cycles of heavy yield (ON crop) one year are followed by a light yield (OFF crop) the next. Heavy yield usually reduces flowering intensity the following year. Despite its agricultural importance, how the developing crop influences the following year''s return bloom and yield is not fully understood. It might be assumed that an ‘AB signal’ is generated in the fruit, or in another organ that senses fruit presence, and moves into the bud to determine its fate—flowering or vegetative growth. The bud then responds to fruit presence by altering regulatory and metabolic pathways. Determining these pathways, and when they are altered, might indicate the nature of this putative AB signal. We studied bud morphology, the expression of flowering control genes, and global gene expression in ON- and OFF-crop buds. In May, shortly after flowering and fruit set, OFF-crop buds were already significantly longer than ON-crop buds. The number of differentially expressed genes was higher in May than at the other tested time points. Processes differentially expressed between ON- and OFF-crop trees included key metabolic and regulatory pathways, such as photosynthesis and secondary metabolism. The expression of genes of trehalose metabolism and flavonoid metabolism was validated by nCounter technology, and the latter was confirmed by metabolomic analysis. Among genes induced in OFF-crop trees was one homologous to SQUAMOSA PROMOTER BINDING-LIKE (SPL), which controls juvenile-to-adult and annual phase transitions, regulated by miR156. The expression pattern of SPL-like, miR156 and other flowering control genes suggested that fruit load affects bud fate, and therefore development and metabolism, a relatively long time before the flowering induction period. Results shed light on some of the metabolic and regulatory processes that are altered in ON and OFF buds.     

Long-Term Effectiveness of a Lifestyle Intervention for the Primary Prevention of Type 2 Diabetes in a Low Socio-Economic Community – An Intervention Follow-Up Study on Reunion Island

In type 2 diabetes (T2D) prevention research, evidence for maintenance of risk factor reduction after three years of follow-up is needed. The objective of this study was to evaluate the long-term effectiveness of a combined lifestyle intervention aiming at controlling body weight (BW) and waist circumference (WC) in non-diabetic, overweight/obese adults living in a low socio-economic community. On Reunion Island, 445 adults living in deprived areas, aged 18–40 and at high-risk for T2D, were included in an intervention versus control trial for primary prevention (2001–2002). The intervention promoted a healthy diet and moderate regular physical activity, through actions strengthening individuals or community and improving living conditions. The control group received a one-shot medical information and nutritional advices. After the end of the trial (2003), 259 of the subjects participated in a follow-up study (2010–2011). The outcomes were the nine-year changes from baseline in BW, body mass index (BMI) and WC measurements, separately. Statistical analyses were performed on an intention-to-treat basis, using available and imputed datasets. At inclusion, T2D risk factors were prevalent: family history of diabetes in first-degree relatives (42%), women with a personal history of gestational diabetes (11%), total obesity (43%, median BMI 29.1 kg/m²) and central obesity (71%). At follow-up, the adjusted effect on imputed dataset was significant for WC -2.4 cm (95% confidence interval: -4.7 to -0.0 cm, p = 0.046), non-significant for BW -2.2 kg (-4.6 to +0.2 kg, p = 0.073) and BMI -0.81 kg/m² (-1.69 to +0.08 kg/m², p = 0.074). A specific long-term effect was the increased likelihood of reduction in adiposity: BW loss, BMI reduction, and WC reduction were more frequent in the intervention group. In the context of low socio-economic communities, our data support the assumption of long-term effect of lifestyle interventions targeting total obesity and central obesity two major drivers of T2D.     

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.)

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed‐specific overexpression of AtGCHI caused significant increases of up to 150‐fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 μg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.     

Methyl-CpG-binding domain proteins in plants: interpreters of DNA methylation

The effect of DNA methylation on various aspects of plant cellular and developmental processes has been well documented over the past 35 years. However, the underlying molecular mechanism interpreting the methylation signal has only recently been explored with the isolation and characterization of the Arabidopsis methyl-CpG-binding domain (MBD) proteins. In this review, we highlight recent advances and present new models concerning Arabidopsis MBD proteins and their possible role in controlling chromatin structure mediated by CpG methylation.     

The three methyl-CpG-binding domains of AtMBD7 control its subnuclear localization and mobility

Three methyl-CpG-binding domain (MBD) proteins in Arabidopsis, AtMBD5, AtMBD6, and AtMBD7, are functional in binding methylated CpG dinucleotides in vitro and localize to the highly CpG-methylated chromocenters in vivo. These proteins differ, however, in their subnuclear localization pattern; AtMBD5 and AtMBD6, each containing a single MBD motif, show preference for two perinucleolar chromocenters, whereas AtMBD7, a naturally occurring poly-MBD protein containing three MBD motifs, localizes to all chromocenters. Here we studied the significance of multiple MBD motifs for subnuclear localization and mobility in living cells. We found that the number of MBD motifs determines the subnuclear localization of the MBD protein. Furthermore, live kinetic experiments showed that AtMBD7-green fluorescent protein (GFP) has lower mobility than AtMBD5-GFP and AtMBD6-GFP, which is conferred by cooperative activity of its three MBD motifs. Thus, the number of MBD motifs appears to affect not only binding affinity and mobility within the nucleus, but also the subnuclear localization of the protein. Our results suggest that poly-MBD proteins can directly affect chromatin structure by inducing intra- and inter-chromatin compaction via bridging over multiple methylated CpG sites.     

Different domains control the localization and mobility of LIKE HETEROCHROMATIN PROTEIN1 in Arabidopsis nuclei

Plants possess a single gene for the structurally related HETEROCHROMATIN PROTEIN1 (HP1), termed LIKE-HP1 (LHP1). We investigated the subnuclear localization, binding properties, and dynamics of LHP1 proteins in Arabidopsis thaliana cells. Transient expression assays showed that tomato (Solanum lycopersicum) LHP1 fused to green fluorescent protein (GFP; Sl LHP1-GFP) and Arabidopsis LHP1 (At LHP1-GFP) localized to heterochromatic chromocenters and showed punctuated distribution within the nucleus; tomato but not Arabidopsis LHP1 was also localized within the nucleolus. Mutations of aromatic cage residues that recognize methyl K9 of histone H3 abolished their punctuated distribution and localization to chromocenters. Sl LHP1-GFP plants displayed cell type-dependent subnuclear localization. The diverse localization pattern of tomato LHP1 did not require the chromo shadow domain (CSD), whereas the chromodomain alone was insufficient for localization to chromocenters; a nucleolar localization signal was identified within the hinge region. Fluorescence recovery after photobleaching showed that Sl LHP1 is a highly mobile protein whose localization and retention are controlled by distinct domains; retention at the nucleolus and chromocenters is conferred by the CSD. Our results imply that LHP1 recruitment to chromatin is mediated, at least in part, through interaction with methyl K9 and that LHP1 controls different nuclear processes via transient binding to its nuclear sites.