An inosine triphosphate pyrophosphatase safeguards plant nucleic acids from aberrant purine nucleotides

In plants, inosine is enzymatically introduced in some tRNAs, but not in other RNAs or DNA. Nonetheless, our data show that RNA and DNA from Arabidopsis thaliana contain (deoxy)inosine, probably derived from nonenzymatic adenosine deamination in nucleic acids and usage of (deoxy)inosine triphosphate (dITP and ITP) during nucleic acid synthesis. We combined biochemical approaches, LC–MS, as well as RNA-Seq to characterize a plant INOSINE TRIPHOSPHATE PYROPHOSPHATASE (ITPA) from A. thaliana, which is conserved in many organisms, and investigated the sources of deaminated purine nucleotides in plants. Inosine triphosphate pyrophosphatase dephosphorylates deaminated nucleoside di- and triphosphates to the respective monophosphates. ITPA loss-of-function causes inosine di- and triphosphate accumulation in vivo and an elevated inosine and deoxyinosine content in RNA and DNA, respectively, as well as salicylic acid (SA) accumulation, early senescence, and upregulation of transcripts associated with immunity and senescence. Cadmium-induced oxidative stress and biochemical inhibition of the INOSINE MONOPHOSPHATE DEHYDROGENASE leads to more IDP and ITP in the wild-type (WT), and this effect is enhanced in itpa mutants, suggesting that ITP originates from ATP deamination and IMP phosphorylation. Inosine triphosphate pyrophosphatase is part of a molecular protection system in plants, preventing the accumulation of (d)ITP and its usage for nucleic acid synthesis

Expression analysis of candidate genes as indicators for commencing drought stress in starch potatoes

Drought stress is a major problem for potato production and will be of grave importance due to climate change and the resulting temperature peaks along with drought periods in the vegetative growth phase of potato. Plants, as sessile organisms, adapt to their environment morphologically as well as biochemically. To cope better with abiotic stresses like drought, plants developed strategies like reactive oxygen species (ROS) detoxification and fast reacting stomatal closure, as well as signalling cascades leading to a quick response to stress. This study aimed at analysing eight genes of interest, derived from a former proteomic study, and determining their suitability for detection of commencing drought stress in early growth stages of potato. For this aim, six starch potato genotypes, which differed in stress response in previous studies, were examined for plant growth and physiological parameters in two experiments in an open greenhouse after seven and 14 days of stress. Besides lower shoot biomass after drought stress, which was already visible after seven days and became stronger after 14 days, weaker root growth was also detected after 14 days. The observed differences between the experiments can presumably be explained by temperature peaks and high radiation prior to and during the first experiment, which took place earlier in the year. The expression of the eight genes was studied in young leaves of four genotypes after 7 days of water withdrawal. Gene expression patterns were dependent on the studied genes. Three genes, cell wall/vacuolar inhibitor of fructosidase (INH1), peroxidase 51-like (POD) and subtilase family protein (SBT1.7) showed consistent changes in gene expression after seven days of stress between all genotypes. The INH1 gene was found to be upregulated in all genotypes in two independent experiments after drought stress. This correlates with the results at the protein level, where INH1 was also found to be higher abundant in two genotypes of potato (Wellpott et al., DGG-Proceedings 10, 2021). Therefore, this gene might be an appropriate candidate for the detection of commencing drought stress in potato

Russeting in Apple Is Initiated After Exposure to Moisture Ends—I. Histological Evidence

Russeting (periderm formation) is a critical fruit-surface disorder in apple (Malus × domestica Borkh.). The first symptom of insipient russeting is cuticular microcracking. Humid and rainy weather increases russeting. The aim was to determine the ontogeny of moisture-induced russeting in ‘Pinova’ apple. We recorded the effects of duration of exposure to water and the stage of fruit development at exposure on microcracking, periderm formation and cuticle deposition. Early on (21 or 31 days after full bloom; DAFB) short periods (2 to 12 d) of moisture exposure induced cuticular microcracking—but not later on (66 or 93 DAFB). A periderm was not formed during moisture exposure but 4 d after exposure ended. A periderm was formed in the hypodermis beneath a microcrack. Russeting frequency and severity were low for up to 4 d of moisture exposure but increased after 6 d. Cuticle thickness was not affected by moisture for up to 8 d but decreased for longer exposures. Cuticular ridge thickness decreased around a microcrack. In general, moisture did not affect cuticular strain release. We conclude that a hypodermal periderm forms after termination of moisture exposure and after microcrack formation. Reduced cuticle deposition may cause moisture-induced microcracking and, thus, russeting. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Time course of changes in the transcriptome during russet induction in apple fruit

Background: Russeting is a major problem in many fruit crops. Russeting is caused by environmental factors such as wounding or moisture exposure of the fruit surface. Despite extensive research, the molecular sequence that triggers russet initiation remains unclear. Here, we present high-resolution transcriptomic data by controlled russet induction at very early stages of fruit development. During Phase I, a patch of the fruit surface is exposed to surface moisture. For Phase II, moisture exposure is terminated, and the formerly exposed surface remains dry. We targeted differentially expressed transcripts as soon as 24 h after russet induction. Results: During moisture exposure (Phase I) of ‘Pinova’ apple, transcripts associated with the cell cycle, cell wall, and cuticle synthesis (SHN3) decrease, while those related to abiotic stress increase. NAC35 and MYB17 were the earliest induced genes during Phase I. They are therefore linked to the initial processes of cuticle microcracking. After moisture removal (Phase II), the expression of genes related to meristematic activity increased (WOX4 within 24 h, MYB84 within 48 h). Genes related to lignin synthesis (MYB52) and suberin synthesis (MYB93, WRKY56) were upregulated within 3 d after moisture removal. WOX4 and AP2B3 are the earliest differentially expressed genes induced in Phase II. They are therefore linked to early events in periderm formation. The expression profiles were consistent between two different seasons and mirrored differences in russet susceptibility in a comparison of cultivars. Furthermore, expression profiles during Phase II of moisture induction were largely identical to those following wounding. Conclusions: The combination of a unique controlled russet induction technique with high-resolution transcriptomic data allowed for the very first time to analyse the formation of cuticular microcracks and periderm in apple fruit immediately after the onset of triggering factors. This data provides valuable insights into the spatial-temporal dynamics of russeting, including the synthesis of cuticles, dedifferentiation of cells, and impregnation of cell walls with suberin and lignin

Apple fruit periderms (russeting) induced by wounding or by moisture have the same histologies, chemistries and gene expressions

Russeting is a cosmetic defect of some fruit skins. Russeting (botanically: induction of periderm formation) can result from various environmental factors including wounding and surface moisture. The objective was to compare periderms resulting from wounding with those from exposure to moisture in developing apple fruit. Wounding or moisture exposure both resulted in cuticular microcracking. Cross-sections revealed suberized hypodermal cell walls by 4 d, and the start of periderm formation by 8 d after wounding or moisture treatment. The expression of selected target genes was similar in wound and moisture induced periderms. Transcription factors involved in the regulation of suberin (MYB93) and lignin (MYB42) synthesis, genes involved in the synthesis (CYP86B1) and the transport (ABCG20) of suberin monomers and two uncharacterized transcription factors (NAC038 and NAC058) were all upregulated in induced periderm samples. Genes involved in cutin (GPAT6, SHN3) and wax synthesis (KCS10, WSD1, CER6) and transport of cutin monomers and wax components (ABCG11) were all downregulated. Levels of typical suberin monomers (ω-hydroxy-C20, -C22 and -C24 acids) and total suberin were high in the periderms, but low in the cuticle. Periderms were induced only when wounding occurred during early fruit development (32 and 66 days after full bloom (DAFB)) but not later (93 DAFB). Wound and moisture induced periderms are very similar morphologically, histologically, compositionally and molecularly

Russeting in apple is initiated after exposure to moisture ends: Molecular and biochemical evidence

Exposure of the fruit surface to moisture during early development is causal in russeting of apple (Malus × domestica Borkh.). Moisture exposure results in formation of microcracks and de-creased cuticle thickness. Periderm differentiation begins in the hypodermis, but only after discon-tinuation of moisture exposure. Expressions of selected genes involved in cutin, wax and suberin synthesis were quantified, as were the wax, cutin and suberin compositions. Experiments were con-ducted in two phases. In Phase I (31 days after full bloom) the fruit surface was exposed to moisture for 6 or 12 d. Phase II was after moisture exposure had been discontinued. Unexposed areas on the same fruit served as unexposed controls. During Phase I, cutin and wax synthesis genes were down-regulated only in the moisture-exposed patches. During Phase II, suberin synthesis genes were up-regulated only in the moisture-exposed patches. The expressions of cutin and wax genes in the moisture-exposed patches increased slightly during Phase II, but the levels of expression were much lower than in the control patches. Amounts and compositions of cutin, wax and suberin were con-sistent with the gene expressions. Thus, moisture-induced russet is a two-step process: moisture exposure reduces cutin and wax synthesis, moisture removal triggers suberin synthesis. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Transcriptomic and functional genomics of russeting on apple

Russeting is a common, economically important fruit skin disorder that occurs in many fruit crops, including apples (Malus x domestica, Borkh.). This skin disorder is caused by environmental factors such as high humidity, prolonged periods of surface moisture, and wounding. While previous studies have focused on the later stages of russeting, the initiation sequence of this disorder remains unclear. To address this knowledge gap, this dissertation aimed to: (1) establish an induction system for russeting in apple fruit using prolonged periods of surface moisture, (2) determine the sequence of russet formation on a histological level under prolonged periods of surface moisture, (3) investigate the behavior of candidate genes involved in periderm and cuticle-related processes on the gene expression as well as metabolic changes, (4) compare the sequence of wound- and moisture-induced russeting, (5) provide a transcriptomic resource for the initial processes during russet formation in apples, and (6) to provide preliminary data on potential candidate genes involved in russeting. Application of surface moisture using a fixed polyethylene tube to developing apple fruit was found to induce microcracks in the fruit skin during early fruit development, and an increase in water vapor permeance was observed in microcracked compared to non-microcracked fruit skins. Microcracked fruit surfaces developed russeting. Histological, gene expression, and metabolic analyses revealed a biphasic behavior during the formation of russeting in response to surface moisture. In Phase I, microcracks appeared within 2 d of moisture treatment and expanded over time, accompanied by a decrease in cuticle-related genes and cutin- and wax-specific metabolites. In Phase II, microcracking decreased after moisture removal, and periderm formation was observed starting 4 d after moisture removal, accompanied by an increase in periderm related genes and suberin specific metabolites. Russeting was observed during Phase II only after at least 6 d of moisture exposure in Phase I. Histological, gene expression, and metabolic analyses showed that the sequence of russeting initiation during Phase II induced by surface moisture was similar to that induced by skin wounding. Transcriptomic analyses revealed that Phase I was characterized by a decrease in cell cycle, cell wall, and cuticle-related genes and an increase in stress-related genes, whereas Phase II was characterized by an increase in meristematic activity, followed by an increase in abscisic acid, lignin, and suberin-related genes. Overall, the studies of this dissertation provide for the first time information on the initial processes of russeting in apple fruit skin and are a valuable resource for future research on the molecular mechanisms underlying this phenomenon

Time course of changes in the transcriptome during russet induction in apple fruit

Abstract Background Russeting is a major problem in many fruit crops. Russeting is caused by environmental factors such as wounding or moisture exposure of the fruit surface. Despite extensive research, the molecular sequence that triggers russet initiation remains unclear. Here, we present high-resolution transcriptomic data by controlled russet induction at very early stages of fruit development. During Phase I, a patch of the fruit surface is exposed to surface moisture. For Phase II, moisture exposure is terminated, and the formerly exposed surface remains dry. We targeted differentially expressed transcripts as soon as 24 h after russet induction. Results During moisture exposure (Phase I) of ‘Pinova’ apple, transcripts associated with the cell cycle, cell wall, and cuticle synthesis (SHN3) decrease, while those related to abiotic stress increase. NAC35 and MYB17 were the earliest induced genes during Phase I. They are therefore linked to the initial processes of cuticle microcracking. After moisture removal (Phase II), the expression of genes related to meristematic activity increased (WOX4 within 24 h, MYB84 within 48 h). Genes related to lignin synthesis (MYB52) and suberin synthesis (MYB93, WRKY56) were upregulated within 3 d after moisture removal. WOX4 and AP2B3 are the earliest differentially expressed genes induced in Phase II. They are therefore linked to early events in periderm formation. The expression profiles were consistent between two different seasons and mirrored differences in russet susceptibility in a comparison of cultivars. Furthermore, expression profiles during Phase II of moisture induction were largely identical to those following wounding. Conclusions The combination of a unique controlled russet induction technique with high-resolution transcriptomic data allowed for the very first time to analyse the formation of cuticular microcracks and periderm in apple fruit immediately after the onset of triggering factors. This data provides valuable insights into the spatial-temporal dynamics of russeting, including the synthesis of cuticles, dedifferentiation of cells, and impregnation of cell walls with suberin and lignin