Specialized replication mechanisms maintain genome stability at human centromeres.

The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.

CENP-B-mediated DNA loops regulate activity and stability of human centromeres.

Chromosome inheritance depends on centromeres, epigenetically specified regions of chromosomes. While conventional human centromeres are known to be built of long tandem DNA repeats, much of their architecture remains unknown. Using single-molecule techniques such as AFM, nanopores, and optical tweezers, we find that human centromeric DNA exhibits complex DNA folds such as local hairpins. Upon binding to a specific sequence within centromeric regions, the DNA-binding protein CENP-B compacts centromeres by forming pronounced DNA loops between the repeats, which favor inter-chromosomal centromere compaction and clustering. This DNA-loop-mediated organization of centromeric chromatin participates in maintaining centromere position and integrity upon microtubule pulling during mitosis. Our findings emphasize the importance of DNA topology in centromeric regulation and stability.

A genetic memory initiates the epigenetic loop necessary to preserve centromere position.

Centromeres are built on repetitive DNA sequences (CenDNA) and a specific chromatin enriched with the histone H3 variant CENP-A, the epigenetic mark that identifies centromere position. Here, we interrogate the importance of CenDNA in centromere specification by developing a system to rapidly remove and reactivate CENP-A (CENP-AOFF/ON ). Using this system, we define the temporal cascade of events necessary to maintain centromere position. We unveil that CENP-B bound to CenDNA provides memory for maintenance on human centromeres by promoting de novo CENP-A deposition. Indeed, lack of CENP-B favors neocentromere formation under selective pressure. Occasionally, CENP-B triggers centromere re-activation initiated by CENP-C, but not CENP-A, recruitment at both ectopic and native centromeres. This is then sufficient to initiate the CENP-A-based epigenetic loop. Finally, we identify a population of CENP-A-negative, CENP-B/C-positive resting CD4+ T cells capable to re-express and reassembles CENP-A upon cell cycle entry, demonstrating the physiological importance of the genetic memory.

Human chromosome-specific aneuploidy is influenced by DNA-dependent centromeric features.

Intrinsic genomic features of individual chromosomes can contribute to chromosome-specific aneuploidy. Centromeres are key elements for the maintenance of chromosome segregation fidelity via a specialized chromatin marked by CENP-A wrapped by repetitive DNA. These long stretches of repetitive DNA vary in length among human chromosomes. Using CENP-A genetic inactivation in human cells, we directly interrogate if differences in the centromere length reflect the heterogeneity of centromeric DNA-dependent features and whether this, in turn, affects the genesis of chromosome-specific aneuploidy. Using three distinct approaches, we show that mis-segregation rates vary among different chromosomes under conditions that compromise centromere function. Whole-genome sequencing and centromere mapping combined with cytogenetic analysis, small molecule inhibitors, and genetic manipulation revealed that inter-chromosomal heterogeneity of centromeric features, but not centromere length, influences chromosome segregation fidelity. We conclude that faithful chromosome segregation for most of human chromosomes is biased in favor of centromeres with high abundance of DNA-dependent centromeric components. These inter-chromosomal differences in centromere features can translate into non-random aneuploidy, a hallmark of cancer and genetic diseases.

CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy.

Chromosome segregation relies on centromeres, yet their repetitive DNA is often prone to aberrant rearrangements under pathological conditions. Factors that maintain centromere integrity to prevent centromere-associated chromosome translocations are unknown. Here, we demonstrate the importance of the centromere-specific histone H3 variant CENP-A in safeguarding DNA replication of alpha-satellite repeats to prevent structural aneuploidy. Rapid removal of CENP-A in S phase, but not other cell-cycle stages, caused accumulation of R loops with increased centromeric transcripts, and interfered with replication fork progression. Replication without CENP-A causes recombination at alpha-satellites in an R loop-dependent manner, unfinished replication, and anaphase bridges. In turn, chromosome breakage and translocations arise specifically at centromeric regions. Our findings provide insights into how specialized centromeric chromatin maintains the integrity of transcribed noncoding repetitive DNA during S phase.

Dispensers for pheromonal pest control.

The detrimental effects of pesticides on the environment and human health have motivated the development of alternative pest control strategies. Pheromonal pest control is one alternative strategy that is attractive because most pheromones used commercially are non-toxic. Pheromones are also effective at low concentrations, and insects are slower to develop resistance to them compared to pesticides. Pheromones can be used to control pests by attracting them towards traps, repelling them from crops, or disrupting their mating behaviour. Viability of pheromonal control strategies must be evaluated on a case-by-case basis and depends on the target species, the pheromone being used, the specific control strategy, the method of dispensing pheromone, other pest control strategies pheromones being used alongside, and many other factors. The efficacy of pheromonal control has been demonstrated in commercial applications such as the control of palm weevils using traps releasing their male aggregation pheromone. Mating disruption using female sex pheromones has also been widely applied for control of both the codling moth Cydia Pomonella and the european grapevine moth Lobesia Botrana (Bangels and Beliën, 2012; Lucchi et al., 2018). Pheromones are volatiles that both degrade quickly in the environment and can be rapidly dispersed by wind. Consequently, administering pheromones to fields requires the use of dispensers that emits pheromone continuously or intermittently. Septum dispensers, membrane dispensers and solid matrix dispensers are best suited to treating smaller areas of cropland since they need to be installed by hand, a labor-intensive process. For treating a large area with pheromones, sprayable formulations and aerosol dispensers are alternative dispensing technologies that can be employed. The characteristics of these different dispenser designs are discussed as well as the kinetics governing pheromone release. Possible areas for future work in pheromone dispenser technology include examining new integrated strategies that employ pheromones alongside other pest control techniques in unique ways. The combination of pheromonal control with physical exclusion or predator release are examples of integrated strategies that are promising but have yet to be widely commercialized. Most commercial pheromonal dispensers are also noted to be impossible or impractical to reuse, apart from aerosol devices. Creating new types of rechargeable dispenser might have some cost saving benefits and would be an interesting area for future innovation in this field.

Solid-state co-culture fermentation of simulated food waste with filamentous fungi for production of bio-pigments.

The use of waste stream residues as feedstock for material production simultaneously helps reduce dependence on fossil-based resources and to shift toward a circular economy. This study explores the conversion of food waste into valuable chemicals, namely, bio-pigments. Here, a simulated food waste feedstock was converted into pigments via solid-state fermentation with the filamentous fungus Talaromyces albobiverticillius (NRRL 2120). Pigments including monascorubrin, rubropunctatin, and 7-(2-hydroxyethyl)-monascorubramine were identified as products of the fermentation via ultra-performance liquid chromatography coupled with quadrupole-time-of-flight electrospray ionization mass spectrometry. Pigments were obtained at concentrations of 32.5, 20.9, and 22.4 AU/gram dry substrate for pigments absorbing at 400, 475, and 500 nm, respectively. Pigment production was further enhanced by co-culturing T. albobiverticillius with Trichoderma reesei (NRRL 3652), and ultimately yielded 63.8, 35.6, and 43.6 AU/gds at the same respective wavelengths. This represents the highest reported production of pigments via solid-state fermentation of a non-supplemented waste stream feedstock. KEY POINTS: • Simulated food waste underwent solid-state fermentation via filamentous fungi. • Bio-pigments were obtained from fermentation of the simulated food waste. • Co-culturing multiple fungal species substantially improved pigment production.

Development of a Nuclear Magnetic Resonance Method and a Near Infrared Calibration Model for the Rapid Determination of Lipid Content in the Field Pea (Pisum sativum).

Pisum sativum is a leguminous crop suitable for cultivation worldwide. It is used as a forage or dried seed supplement in animal feed and, more recently, as a potential non-traditional oilseed. This study aimed to develop a low-cost, rapid, and non-destructive method for analyzing pea lipids with no chemical modifications that would prove superior to existing destructive solvent extraction methods. Different pea accession seed samples, prepared as either small portions (0.5 mm2) of endosperm or ground pea seed powder for comparison, were subjected to HR-MAS NMR analyses and whole seed samples underwent NIR analyses. The total lipid content ranged between 0.57-3.45% and 1.3-2.6% with NMR and NIR, respectively. Compared to traditional extraction with butanol, hexane-isopropanol, and petroleum ether, correlation coefficients were 0.77 (R2 = 0.60), 0.56 (R2 = 0.47), and 0.78 (R2 = 0.62), respectively. Correlation coefficients for NMR compared to traditional extraction increased to 0.97 (R2 = 0.99) with appropriate correction factors. PLS regression analyses confirmed the application of this technology for rapid lipid content determination, with trends fitting models often close to an R2 of 0.95. A better robust NIR quantification model can be developed by increasing the number of samples with more diversity.

Plk1 protects kinetochore-centromere architecture against microtubule pulling forces.

During mitosis, sister chromatids attach to microtubules which generate ~ 700 pN pulling force focused on the centromere. We report that chromatin-localized signals generated by Polo-like kinase 1 (Plk1) maintain the integrity of the kinetochore and centromere against this force. Without sufficient Plk1 activity, chromosomes become misaligned after normal condensation and congression. These chromosomes are silent to the mitotic checkpoint, and many lag and mis-segregate in anaphase. Their centromeres and kinetochores lack CENP-A, CENP-C, CENP-T, Hec1, Nuf2, and Knl1; however, CENP-B is retained. CENP-A loss occurs coincident with secondary misalignment and anaphase onset. This disruption occurs asymmetrically prior to anaphase and requires tension generated by microtubules. Mechanistically, centromeres highly recruit PICH DNA helicase and PICH depletion restores kinetochore disruption in pre-anaphase cells. Furthermore, anaphase defects are significantly reduced by tethering Plk1 to chromatin, including H2B, and INCENP, but not to CENP-A. Taken as a whole, this demonstrates that Plk1 signals are crucial for stabilizing centromeric architecture against tension.

Chronic Granulomatous Disease with the McLeod Phenotype: a French National Retrospective Case Series.

BACKGROUND: X-linked chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the CYBB gene (located on Xp21.1). Patients with large deletions on chromosome Xp21.1 can present with the McLeod phenotype and also Duchenne muscular dystrophy or retinitis pigmentosa. The objective of the present study was to describe a series of French patients with CGD and the McLeod phenotype. METHODS: We retrospectively collected data from the medical records of 8 patients with CGD and the McLeod phenotype registered at the French National Reference Center for blood types. RESULTS: The median age at diagnosis of CGD was 1.2 years, the median age at diagnosis of the McLeod phenotype was 4.5 years, and the median length of follow-up was 15.2 years. Four patients displayed allo-immunization, with anti-KEL20 and anti-XK1 (formerly known as anti-KL) antibodies. Five of the 6 patients with available blood smears had acanthocytosis. Neuropsychiatric, muscle-related, and ocular manifestations were present in 4, 2, and 1 of the patients, respectively. Three of the 4 patients having undergone allogeneic hematopoietic stem cell transplantation (HSCT) are alive. Overall, 5 patients are alive, and 3 are alive and well. CONCLUSION: This is the largest yet descriptive study of a series of patients with X-linked CGD and the McLeod phenotype. Although this disease combination is rare, the timely, accurate diagnosis of the McLeod phenotype is critical because of the serious post-transfusion complications. However, HSCT can be considered in these patients.

Home versus laboratory assessments of melatonin production and melatonin onset in young adults complaining of a delayed sleep schedule.

Recent evidence points toward an association between higher non-visual sensitivity to light and a later circadian phase in young adults complaining of a delayed sleep schedule. Light exposure in the evening may therefore induce a larger suppression of melatonin production in these individuals, which might: (a) bias home estimates of melatonin onset; and (b) decrease sleep propensity at bedtime. In this study, we compared home and laboratory melatonin onsets and production in sleep-delayed and control participants, using saliva samples collected in the 3 hr preceding habitual bedtime. The mean light intensity measured during saliva sampling at home was ~10 lux in both groups. Melatonin suppression at home was significant, averaging 31% and 24% in sleep-delayed and control individuals, respectively. Group difference in melatonin suppression was not significant. Estimates of melatonin onset were on average 27 min later at home than in laboratory conditions, with no group difference. Looking specifically at sleep-delayed participants, there was no correlation between non-visual sensitivity to light and home-laboratory differences in melatonin onsets. However, higher light sensitivity was associated with greater melatonin suppression in the hour before habitual bedtime. Greater melatonin suppression before bedtime was also associated with a later circadian phase. These results indicate that the validity of home estimates of melatonin onset is similar in sleep-delayed and in control individuals. Results also suggest that increased non-visual sensitivity to light could impact melatonin secretion in sleep-delayed individuals and contribute to a late bedtime by delaying circadian phase and decreasing sleep propensity.

Efficient Enzymatic Hydrolysis of Biomass Hemicellulose in the Absence of Bulk Water.

Current enzymatic methods for hemicellulosic biomass depolymerization are solution-based, typically require a harsh chemical pre-treatment of the material and large volumes of water, yet lack in efficiency. In our study, xylanase (E.C. 3.2.1.8) from Thermomyces lanuginosus is used to hydrolyze xylans from different sources. We report an innovative enzymatic process which avoids the use of bulk aqueous, organic or inorganic solvent, and enables hydrolysis of hemicellulose directly from chemically untreated biomass, to low-weight, soluble oligoxylosaccharides in >70% yields.

Model Study To Assess Softwood Hemicellulose Hydrolysates as the Carbon Source for PHB Production in Paraburkholderia sacchari IPT 101.

Softwood hemicellulose hydrolysates are a cheap source of sugars that can be used as a feedstock to produce polyhydroxybutyrates (PHB), which are biobased and compostable bacterial polyesters. To assess the potential of the hemicellulosic sugars as a carbon source for PHB production, synthetic media containing softwood hemicellulose sugars (glucose, mannose, galactose, xylose, arabinose) and the potentially inhibitory lignocellulose degradation products (acetic acid, 5-hydroxymethylfurfural (HMF), furfural, and vanillin) were fermented with the model strain Paraburkholderia sacchari IPT 101. Relative to pure glucose, individual fermentation for 24 h with 20 g/L mannose or galactose exhibited maximum specific growth rates of 97% and 60%, respectively. On the other hand, with sugar mixtures of glucose, mannose, galactose, xylose, and arabinose, the strain converted all sugars simultaneously to reach a maximum PHB concentration of 5.72 g/L and 80.5% PHB after 51 h. The addition of the inhibitor mixture at the following concentration, sodium acetate (2.11 g/L), HMF (0.67 g/L), furfural (0.66 g/L), and vanillin (0.93 g/L), to the sugar mixture stopped the growth entirely within 24 h. Individually, the inhibitors either had no effect or only reduced growth. Moreover, it was found that a bacterial inoculum with high initial cell density (optical density, OD ≥ 5.6) could overcome the growth inhibition to yield an OD of 13 within 24 h. Therefore, softwood hemicellulose sugars are viable carbon sources for PHB production. Nevertheless, real softwood hemicellulose hydrolysates need detoxification or a high inoculum to overcome inhibitory effects and allow bacterial growth.

Sex Differences in Stroke Attack, Incidence, and Mortality Rates in Northern France.

BACKGROUND: Age and sex have a major impact on stroke onset. AIMS: We aimed to compare the attack, incidence, and 28-day mortality rate for stroke as well as risk factors in men and women aged 35 and over. METHODS: Data were obtained between 2008 and 2015 from the stroke population-based registry covering the city of Lille (northern France). RESULTS: A total of 2426 strokes (1917 incident strokes) were recorded. The number of strokes was lower in women than in men when considering individuals under the age of 75 but was twice as high when considering individuals aged 75 or over. Overall, there were 25% more strokes in women than in men. The age-adjusted attack (P = .017) and incident (P = .027) rates of stroke were ~30% lower in women than in men (a ~30% lower risk of ischemic stroke (P = .02) and a ~40% lower risk of intracerebral hemorrhage (ICH) (P = .004)). The age-adjusted mortality rate after ICH was ~35% lower in women than in men (P = .014). With regard to cardiovascular risk factors, women with stroke were older, smoked less, and were more likely to have a history of migraine or atrial fibrillation than the men. CONCLUSION: The risk of stroke is lower in women than in men under the age of 75 but is similar when comparing women and men after that age. Nevertheless, the age structure of the population (with more elderly women than elderly men) translates into a higher absolute number of strokes in women than in men.

Plant cell wall imaging by metabolic click-mediated labelling of rhamnogalacturonan II using azido 3-deoxy-D-manno-oct-2-ulosonic acid.

In plants, 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) is a monosaccharide that is only found in the cell wall pectin, rhamnogalacturonan-II (RG-II). Incubation of 4-day-old light-grown Arabidopsis seedlings or tobacco BY-2 cells with 8-azido 8-deoxy Kdo (Kdo-N3 ) followed by coupling to an alkyne-containing fluorescent probe resulted in the specific in muro labelling of RG-II through a copper-catalysed azide-alkyne cycloaddition reaction. CMP-Kdo synthetase inhibition and competition assays showing that Kdo and D-Ara, a precursor of Kdo, but not L-Ara, inhibit incorporation of Kdo-N3 demonstrated that incorporation of Kdo-N3 occurs in RG-II through the endogenous biosynthetic machinery of the cell. Co-localisation of Kdo-N3 labelling with the cellulose-binding dye calcofluor white demonstrated that RG-II exists throughout the primary cell wall. Additionally, after incubating plants with Kdo-N3 and an alkynated derivative of L-fucose that incorporates into rhamnogalacturonan I, co-localised fluorescence was observed in the cell wall in the elongation zone of the root. Finally, pulse labelling experiments demonstrated that metabolic click-mediated labelling with Kdo-N3 provides an efficient method to study the synthesis and redistribution of RG-II during root growth.

Extraction of gelatin from salmon (Salmo salar) fish skin using trypsin-aided process: optimization by Plackett-Burman and response surface methodological approaches.

Gelatin from salmon (Salmo salar) skin with high molecular weight protein chains (α-chains) was extracted using trypsin-aided process. Response surface methodology was used to optimise the extraction parameters. Yield, hydroxyproline content and protein electrophoretic profile via sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of gelatin were used as responses in the optimization study. The optimum conditions were determined as: trypsin concentration at 1.49 U/g; extraction temperature at 45 °C; and extraction time at 6 h 16 min. This response surface optimized model was significant and produced an experimental value (202.04 ± 8.64%) in good agreement with the predicted value (204.19%). Twofold higher yields of gelatin with high molecular weight protein chains were achieved in the optimized process with trypsin treatment when compared to the process without trypsin.

Inhibition of fucosylation of cell wall components by 2-fluoro 2-deoxy-L-fucose induces defects in root cell elongation.

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2-fluoro 2-l-fucose (2F-Fuc) reduces root growth at micromolar concentrations. The inability of 2F-Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F-Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N-linked glycans is fully inhibited by 10 µm 2F-Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F-Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan-II (RG-II). At low concentrations, 2F-Fuc induced a decrease in RG-II dimerization. Both RG-II dimerization and root growth were partially restored in 2F-Fuc-treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F-Fuc was due to a deficiency of RG-II dimerization. Closer investigation of the 2F-Fuc-induced growth phenotype demonstrated that cell division is not affected by 2F-Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG-II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG-II cross-linking, but that it might also be a signal molecule in the cell wall integrity-sensing mechanism.

Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues.

Evolution of severe sleep-wake cycle disturbances following traumatic brain injury: a case study in both acute and subacute phases post-injury.

BACKGROUND: Sleep-wake disturbances are frequently reported following traumatic brain injury (TBI), but they remain poorly documented in the acute stage of injury. Little is known about their origin and evolution. CASE PRESENTATION: This study presents the case of a patient in the acute phase of a severe TBI. The patient was injured at work when falling 12 m into a mine and was hospitalized in the regular wards of a level I trauma centre. From days 31 to 45 post-injury, once he had reached a level of medical stability and continuous analgosedation had been ceased, his sleep-wake cycle was monitored using actigraphy. Results showed significant sleep-wake disturbances and severe sleep deprivation. Indeed, the patient had an average nighttime sleep efficiency of 32.7 ± 15.4 %, and only an average of 4.8 ± 1.3 h of sleep per 24-h period. After hospital discharge to the rehabilitation centre, where he remained for 5 days, the patient was readmitted to the same neurological unit for paranoid delusions. During his second hospital stay, actigraphy recordings resumed from days 69 to 75 post-injury. A major improvement in his sleep-wake cycle was observed during this second stay, with an average nighttime sleep efficiency of 96.3 ± 0.9 % and an average of 14.1 ± 0.9 h of sleep per 24-h period. CONCLUSION: This study is the first to extensively document sleep-wake disturbances in both the acute and subacute phases of severe TBI. Results show that prolonged sleep deprivation can be observed after TBI, and suggest that the hospital environment only partially contributes to sleep-wake disturbances. Continuous actigraphic monitoring may prove to be a useful clinical tool in the monitoring of patients hospitalized after severe TBI in order to detect severe sleep deprivation requiring intervention. The direct impact of sleep-wake disturbances on physiological and cognitive recovery is not well understood within this population, but is worth investigating and improving.

The cell wall pectic polymer rhamnogalacturonan-II is required for proper pollen tube elongation: implications of a putative sialyltransferase-like protein.

BACKGROUND AND AIMS: Rhamnogalacturonan-II (RG-II) is one of the pectin motifs found in the cell wall of all land plants. It contains sugars such as 2-keto-3-deoxy-d-lyxo-heptulosaric acid (Dha) and 2-keto-3-deoxy-d-manno-octulosonic acid (Kdo), and within the wall RG-II is mostly found as a dimer via a borate diester cross-link. To date, little is known regarding the biosynthesis of this motif. Here, after a brief review of our current knowledge on RG-II structure, biosynthesis and function in plants, this study explores the implications of the presence of a Golgi-localized sialyltransferase-like 2 (SIA2) protein that is possibly involved in the transfer of Dha or Kdo in the RG-II of Arabidopsis thaliana pollen tubes, a fast-growing cell type used as a model for the study of cell elongation. METHODS: Two heterozygous mutant lines of arabidopsis (sia2-1+/- and qrt1 × sia2-2+/-) were investigated. sia2-2+/- was in a quartet1 background and the inserted T-DNA contained the reporter gene ß-glucuronidase (GUS) under the pollen-specific promoter LAT52. Pollen germination and pollen tube phenotype and growth were analysed both in vitro and in vivo by microscopy. KEY RESULTS: Self-pollination of heterozygous lines produced no homozygous plants in the progeny, which may suggest that the mutation could be lethal. Heterozygous mutants displayed a much lower germination rate overall and exhibited a substantial delay in germination (20 h of delay to reach 30 % of pollen grain germination compared with the wild type). In both lines, mutant pollen grains that were able to produce a tube had tubes that were either bursting, abnormal (swollen or dichotomous branching tip) or much shorter compared with wild-type pollen tubes. In vivo, mutant pollen tubes were restricted to the style, whereas the wild-type pollen tubes were detected at the base of the ovary. CONCLUSIONS: This study highlights that the mutation in arabidopsis SIA2 encoding a sialyltransferase-like protein that may transfer Dha or Kdo on the RG-II motif has a dramatic effect on the stability of the pollen tube cell wall.