The interaction between irreversible electroporation therapy (IRE) and embolization material using a validated vegetal model: an experimental study.

PURPOSE: Irreversible electroporation (IRE) is a nonthermal tumor ablation technique that induces cell apoptosis while preserving extracellular architecture. Surgical clips and embolic agents may lie adjacent to, or within, the target lesion. It is unknown to date if IRE causes degradation to the embolic agents or surgical clips that may have adverse effects to patients. We aimed to examine the effects of the IRE on the morphology of various embolic agents and the effects of these agents to the ablation field using a previously validated vegetal model. METHODS: Metallic surgical clips and various metallic and nonmetallic embolic agents were inserted within the center of the tuber ablation field. Additionally, clips were inserted on the edge and outside the ablation field. One tuber was ablated as a control. Ablation settings were based on previous published experiments. Tubers were imaged with magnetic resonance imaging (MRI) 18-24 hours after ablation and the ablated field dimensions were measured. Nonmetallic embolic agents were examined microscopically by the pathologist. RESULTS: Nonmetallic agents did not affect the ablation pattern. Metallic implants, however, caused arcing of the ablation margins. There was no macroscopic or microscopic degradation to the agents after IRE. CONCLUSION: The ablation zone arced in the presence of surgical clips at the edge or outside the ablation margins; therefore, nearby critical structures may be susceptible to the effects of IRE. Furthermore, there was no physical degradation of the embolic agents or surgical clips, and this may have importance when considering IRE ablation of previously embolized lesions in vivo.

Sustained substrate cycles between hexose phosphates and free sugars in phosphate-deficient potato (Solanum tuberosum) cell cultures.

MAIN CONCLUSION: Futile cycling between free sugars and hexose phosphates occurring under phosphate deficiency could be involved in the maintenance of a threshold level of free cellular phosphate to preserve respiratory metabolism. We studied the metabolic response of potato cell cultures growing in Pi sufficient (2.5 mM, +Pi) or deficient (125 µM, -Pi) conditions. Under Pi deficiency, cellular growth was severely affected, however -Pi cells were able to maintain a low but steady level of free Pi. We surveyed the activities of 33 primary metabolic enzymes during the course of a 12 days Pi deficiency period. Our results show that many of these enzymes had higher specific activity in -Pi cells. Among these, we found typical markers of Pi deficiency such as phosphoenolpyruvate phosphatase and phosphoenolpyruvate carboxylase as well as enzymes involved in the biosynthesis of organic acids. Intriguingly, several ATP-consuming enzymes such as hexokinase (HK) and phosphofructokinase also displayed increased activity in -Pi condition. For HK, this was associated with an increase in the steady state of a specific HK polypeptide. Quantification of glycolytic intermediates showed a pronounced decrease in phosphate esters under Pi deficiency. Adenylate levels also decreased in -Pi cells, but the Adenylate Energy Charge was not affected by the treatment. To investigate the significance of HK induction under low Pi, [U-14C]-glucose tracer studies were conducted. We found in vivo evidence of futile cycling between pools of hexose phosphates and free sugars under Pi deficiency. Our study suggests that the futile cycling between hexose phosphates and free sugars which is active under +Pi conditions is sustained under Pi deficiency. The possibility that this process represents a metabolic adaptation to Pi deficiency is discussed with respect to Pi homeostasis in Pi-deficient conditions.

Ablation outcome of irreversible electroporation on potato monitored by impedance spectrum under multi-electrode system.

BACKGROUND: Irreversible electroporation (IRE) therapy relies on pulsed electric fields to non-thermally ablate cancerous tissue. Methods for evaluating IRE ablation in situ are critical to assessing treatment outcome. Analyzing changes in tissue impedance caused by electroporation has been proposed as a method for quantifying IRE ablation. In this paper, we assess the hypothesis that irreversible electroporation ablation outcome can be monitored using the impedance change measured by the electrode pairs not in use, getting more information about the ablation size in different directions. METHODS: Using a square four-electrode configuration, the two diagonal electrodes were used to electroporate potato tissue. Next, the impedance changes, before and after treatment, were measured from different electrode pairs and the impedance information was extracted by fitting the data to an equivalent circuit model. Finally, we correlated the change of impedance from various electrode pairs to the ablation geometry through the use of fitted functions; then these functions were used to predict the ablation size and compared to the numerical simulation results. RESULTS: The change in impedance from the electrodes used to apply pulses is larger and has higher deviation than the other electrode pairs. The ablation size and the change in resistance in the circuit model correlate with various linear functions. The coefficients of determination for the three functions are 0.8121, 0.8188 and 0.8691, respectively, showing satisfactory agreement. The functions can well predict the ablation size under different pulse numbers, and in some directions it did even better than the numerical simulation method, which used different electric field thresholds for different pulse numbers. CONCLUSIONS: The relative change in tissue impedance measured from the non-energized electrodes can be used to assess ablation size during treatment with IRE according to linear functions.

PAMP-responsive ATL gene StRFP1 and its orthologue NbATL60 positively regulate Phytophthora infestans resistance in potato and Nicotiana benthamiana.

Ubiquitination is a post-translational modification that plays a crucial role during the regulation of plant immune signalling. The plant ATL family consists of a large number of putative RING type ubiquitin ligases. We show that potato ATL family gene StRFP1 and its orthologue NbATL60 from N. benthamiana both respond to Phytophthora infestans culture filtrate (CF) and flg22 induction. StRFP1 positively regulates immunity against P. infestans in potato. Ectopic transient expression of StRFP1 or expression of NbATL60 in N. benthamiana also enhances late blight resistance. By contrast, silencing NbATL60 in N. benthamiana reduces late blight resistance and leads to plant growth inhibition. Both StRFP1 and NbATL60 localize to the plasma membrane and intracellular puncta and possess E3 Ligase activity in vitro. Furthermore we demonstrate that the RING finger domain mutants of StRFP1 and NbATL60 lost E3 ligase activity and fail to suppress P. infestans colonization in N. benthamiana, indicating that E3 ligase activity is critical for StRFP1 and NbATL60 to regulate immunity. Overexpression or RNA interference of StRFP1 in transgenic potato led to increased or decreased expression of PTI maker genes (WRKY7, WRKY8, ACRE31 and Pti5) respectively. Similarly silencing of NbATL60 in N. benthamiana decreases expression of these PTI marker genes. Moreover, VIGS of NbATL60 in N. benthamiana did not compromise P. infestans PAMP INF1 or R2/Avr2, R3a/AVR3a, Rx/Cp and Pto/AvrPto triggered cell death. These results indicate that ATL genes StRFP1 and NbATL60 contribute to basal immunity (PTI) in Solanaceous plants.

Novel potato plants with enhanced cadmium resistance and antioxidative defence generated after in vitro cell line selection.

It is of interest to apply plant tissue culture to generate plants resistant to toxic effects of cadmium (Cd) on plant growth. Callus cultures were initiated from leaf explants of micropropagated potato plantlets (Solanum tuberosum L., cv. Iwa) for in vitro selection comprising 18 different Cd treatments varying in Cd exposure timing and duration. Plantlets regenerated from two different lines of Cd-selected calli, L9 and L11, were found to exhibit enhanced resistance to 218 µM Cd compared to control (source plantlets for leaf explants used to initiate callus cultures for Cd resistance). In response to 218 µM Cd, L11 plantlets had lower levels of lipid peroxidation and hydrogen peroxide than control and L9 plantlets. In addition, antioxidative enzyme activities in L11 were generally higher than control. L11 also had a higher level of proline than control.

Comparative Morphology, Transcription, and Proteomics Study Revealing the Key Molecular Mechanism of Camphor on the Potato Tuber Sprouting Effect.

Sprouting regulation in potato tubers is important for improving commercial value and producing new plants. Camphor shows flexible inhibition of tuber sprouting and prolongs the storage period of potato, but its underlying mechanism remains unknown. The results of the present study suggest that camphor inhibition caused bud growth deformities and necrosis, but after moving to more ventilated conditions, new sprouts grew from the bud eye of the tuber. Subsequently, the sucrose and fructose contents as well as polyphenol oxidase (PPO) activity were assessed after camphor inhibition. Transcription and proteomics data from dormancy (D), sprouting (S), camphor inhibition (C), and recovery sprouting (R) samples showed changes in the expression levels of approximately 4000 transcripts, and 700 proteins showed different abundances. KEGG (Kyoto encyclopaedia of genes and genomes) pathway analysis of the transcription levels indicated that phytohormone synthesis and signal transduction play important roles in tuber sprouting. Camphor inhibited these processes, particularly for gibberellic acid, brassinosteroids, and ethylene, leading to dysregulation of physiological processes such as cutin, suberine and wax biosynthesis, fatty acid elongation, phenylpropanoid biosynthesis, and starch and sucrose metabolism, resulting in bud necrosis and prolonged storage periods. The KEGG pathway correlation between transcripts and proteins revealed that terpenoid backbone biosynthesis and plant-pathogen interaction pathways showed significant differences in D vs. S samples, but 13 pathways were remarkably different in the D vs. C groups, as camphor inhibition significantly increased both the transcription levels and protein abundance of pathogenesis-related protein PR-10a (or STH-2), the pathogenesis-related P2-like precursor protein, and the kirola-like protein as compared to sprouting. In recovery sprouting, these genes and proteins were decreased at both the transcriptional level and in protein abundance. It was important to find that the inhibitory effect of camphor on potato tuber sprout was reversible, revealing the action mechanism was similar to resistance to pathogen infection. The present study provides a theoretical basis for the application of camphor in prolonging seed potato storage.

Genetic enhancement of oil content in potato tuber (Solanum tuberosum L.) through an integrated metabolic engineering strategy.

Potato tuber is a high yielding food crop known for its high levels of starch accumulation but only negligible levels of triacylglycerol (TAG). In this study, we evaluated the potential for lipid production in potato tubers by simultaneously introducing three transgenes, including WRINKLED 1 (WRI1), DIACYLGLYCEROL ACYLTRANSFERASE 1 (DGAT1) and OLEOSIN under the transcriptional control of tuber-specific (patatin) and constitutive (CaMV-35S) promoters. This coordinated metabolic engineering approach resulted in over a 100-fold increase in TAG accumulation to levels up to 3.3% of tuber dry weight (DW). Phospholipids and galactolipids were also found to be significantly increased in the potato tuber. The increase of lipids in these transgenic tubers was accompanied by a significant reduction in starch content and an increase in soluble sugars. Microscopic examination revealed that starch granules in the transgenic tubers had more irregular shapes and surface indentations when compared with the relatively smooth surfaces of wild-type starch granules. Ultrastructural examination of lipid droplets showed their close proximity to endoplasmic reticulum and mitochondria, which may indicate a dynamic interaction with these organelles during the processes of lipid biosynthesis and turnover. Increases in lipid levels were also observed in the transgenic potato leaves, likely due to the constitutive expression of DGAT1 and incomplete tuber specificity of the patatin promoter. This study represents an important proof-of-concept demonstration of oil increase in tubers and provides a model system to further study carbon reallocation during development of nonphotosynthetic underground storage organs.

The Double-Reduction Landscape in Tetraploid Potato as Revealed by a High-Density Linkage Map.

The creation of genetic linkage maps in polyploid species has been a long-standing problem for which various approaches have been proposed. In the case of autopolyploids, a commonly used simplification is that random bivalents form during meiosis. This leads to relatively straightforward estimation of recombination frequencies using maximum likelihood, from which a genetic map can be derived. However, autopolyploids such as tetraploid potato (Solanum tuberosum L.) may exhibit additional features, such as double reduction, not normally encountered in diploid or allopolyploid species. In this study, we produced a high-density linkage map of tetraploid potato and used it to identify regions of double reduction in a biparental mapping population. The frequency of multivalents required to produce this degree of double reduction was determined through simulation. We also determined the effect that multivalents or preferential pairing between homologous chromosomes has on linkage mapping. Low levels of multivalents or preferential pairing do not adversely affect map construction when highly informative marker types and phases are used. We reveal the double-reduction landscape in tetraploid potato, clearly showing that this phenomenon increases with distance from the centromeres.

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida.

BACKGROUND: The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure - the syncytium - which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium. RESULTS: The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure. CONCLUSION: This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Identification and characterisation of a hyper-variable apoplastic effector gene family of the potato cyst nematodes.

Sedentary endoparasitic nematodes are obligate biotrophs that modify host root tissues, using a suite of effector proteins to create and maintain a feeding site that is their sole source of nutrition. Using assumptions about the characteristics of genes involved in plant-nematode biotrophic interactions to inform the identification strategy, we provide a description and characterisation of a novel group of hyper-variable extracellular effectors termed HYP, from the potato cyst nematode Globodera pallida. HYP effectors comprise a large gene family, with a modular structure, and have unparalleled diversity between individuals of the same population: no two nematodes tested had the same genetic complement of HYP effectors. Individuals vary in the number, size, and type of effector subfamilies. HYP effectors are expressed throughout the biotrophic stages in large secretory cells associated with the amphids of parasitic stage nematodes as confirmed by in situ hybridisation. The encoded proteins are secreted into the host roots where they are detectable by immunochemistry in the apoplasm, between the anterior end of the nematode and the feeding site. We have identified HYP effectors in three genera of plant parasitic nematodes capable of infecting a broad range of mono- and dicotyledon crop species. In planta RNAi targeted to all members of the effector family causes a reduction in successful parasitism.

Copy number variation in potato - an asexually propagated autotetraploid species.

Copy number variation (CNV) has been revealed as a significant contributor to the genetic variation in humans. Although CNV has been reported in several model animal and plant species, the presence of CNV and its biological impact in polyploid species has not yet been documented. We conducted a fluorescence in situ hybridization (FISH)-based CNV survey in potato, a vegetatively propagated autotetraploid species (2n = 4x = 48). We conducted FISH analysis using 18 randomly selected potato bacterial artificial chromosome (BAC) clones in a set of 16 potato cultivars with diverse breeding backgrounds. Six BACs (33%) with insert sizes of 137-145 kb were found to be associated with large CNV events detectable at the cytological level. We demonstrate that the large CNVs associated with two specific BACs (RH102I10 and RH83C08) were widespread among potato cultivars developed in North America and Europe. We measured the transcript abundance of four genes associated with the CNV spanned by BAC RH102I10. All four genes displayed a dosage effect in transcription. Although potato is vegetatively propagated, we observed that female gametes lacking the RH102I10-associated CNV were inferior to those with at least one copy of this CNV, indicating that the RH102I10-associated CNV can impact on the growth and development of the potato plants. Our results show that CNV is highly abundant in the potato genome and may play a significant role in genetic variation of this important food crop.

Differential induction of oxylipin pathway in potato and tobacco cells by bacterial and oomycete elicitors.

KEY MESSAGE: Potato and tobacco cells are differentially suited to study oxylipin pathway and elicitor-induced responses. Synthesis of oxylipins via the lipoxygenase (LOX) pathway provides plant cells with an important class of signaling molecules, related to plant stress responses and innate immunity. The aim of this study was to evaluate the induction of LOX pathway in tobacco and potato cells induced by a concentrated culture filtrate (CCF) from Phytophthora infestans and lipopolysaccharide (LPS) from Pectobacterium atrosepticum. Oxylipin activation was evaluated by the measurement of LOX activity and metabolite quantification. The basal levels of oxylipins and fatty acids showed that potato cells contained higher amounts of linoleic (LA), linolenic (LnA) and stearic acids than tobacco cells. The major oxylipin in potato cells, 9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid (9,10,11-THOD), was not detected in tobacco cells. CCF induced a sharp increase of LA and LnA at 8 h in tobacco cells. In contrast they decreased in potato cells. In CCF-treated tobacco cells, colneleic acid increased up to 24 h, colnelenic acid and 9(S)-hydroxyoctadecatrienoic acid (9(S)-HOT) increased up to 16 h. In potato cells, only colneleic acid increased slightly until 16 h. A differential induction of LOX activity was measured in both cells treated by CCF. With LPS treatment, only 9,10,11-THOD accumulation was significantly induced at 16 h in potato cells. Fatty acids were constant in tobacco but decreased in potato cells over the studied time period. These results showed that the two elicitors were differently perceived by the two Solanaceae and that oxylipin pathway is strongly induced in tobacco with the CCF. They also revealed that elicitor-induced responses depended on both cell culture and elicitor.

The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

Comparison of transcript profiles in different life stages of the nematode Globodera pallida under different host potato genotypes.

The potato cyst nematodes (PCNs) Globodera pallida and Globodera rostochiensis are important parasites of potato. PCNs undergo complex biotrophic interactions with their hosts that involve gene expression changes in both the nematode and the host plant. The aim of this study was to determine key genes that are differentially expressed in Globodera pallida life cycle stages and during the initiation of the feeding site in susceptible and partially resistant potato genotypes. For this purpose, two microarray experiments were designed: (i) a comparison of eggs, infective second-stage juveniles (J2s) and sedentary parasitic-stage J2s (SJ2); (ii) a comparison of SJ2s at 8 days after inoculation (DAI) in the susceptible cultivar (Desirée) and two partially resistant lines. The results showed differential expression of G. pallida genes during the stages studied, including previously characterized effectors. In addition, a large number of genes changed their expression between SJ2s in the susceptible cultivar and those infecting partially resistant lines; the number of genes with modified expression was lower when the two partially resistant lines were compared. Moreover, a histopathological study was performed at several time points (7, 14 and 30 DAI) and showed the similarities between both partially resistant lines with a delay and degeneration in the formation of the syncytia in comparison with the susceptible cultivar. Females at 30 DAI in partially resistant lines showed a delay in their development in comparison with those in the susceptible cultivar.

Harmonic optical microscopy and fluorescence lifetime imaging platform for multimodal imaging.

In this work, we proposed and built a multimodal optical setup that extends a commercially available confocal microscope (Olympus VF300) to include nonlinear second harmonic generation (SHG) and third harmonic generation (THG) optical (NLO) microscopy and fluorescence lifetime imaging microscopy (FLIM). We explored all the flexibility offered by this commercial confocal microscope to include the nonlinear microscopy capabilities. The setup allows image acquisition with confocal, brightfield, NLO/multiphoton and FLIM imaging. Simultaneously, two-photon excited fluorescence (TPEF) and SHG are well established in the biomedical imaging area, because one can use the same ultrafast laser and detectors set to acquire both signals simultaneously. Because the integration with FLIM requires a separated modulus, there are fewer reports of TPEF+SHG+FLIM in the literature. The lack of reports of a TPEF+SHG+THG+FLIM system is mainly due to difficulties with THG because the present NLO laser sources generate THG in an UV wavelength range incompatible with microscope optics. In this article, we report the development of an easy-to-operate platform capable to perform two-photon fluorescence (TPFE), SHG, THG, and FLIM using a single 80 MHz femtosecond Ti:sapphire laser source. We described the modifications over the confocal system necessary to implement this integration and verified the presence of SHG and THG signals by several physical evidences. Finally, we demonstrated the use of this integrated system by acquiring images of vegetables and epithelial cancer biological samples.

Two carbon fluxes to reserve starch in potato (Solanum tuberosum L.) tuber cells are closely interconnected but differently modulated by temperature.

Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-¹4C]glucose 1-phosphate, [U-¹4C]sucrose, [U-¹4C]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-¹4C]sucrose plus unlabelled equimolar glucose 1-phosphate. C¹4-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced ¹4C incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 °C but the flux of the sucrose-dependent route strongly increases above 20 °C. Results are confirmed by in vitro experiments using [U-¹4C]glucose 1-phosphate or adenosine-[U-¹4C]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C¹4C-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.

Release of apical dominance in potato tuber is accompanied by programmed cell death in the apical bud meristem.

Potato (Solanum tuberosum) tuber, a swollen underground stem, is used as a model system for the study of dormancy release and sprouting. Natural dormancy release, at room temperature, is initiated by tuber apical bud meristem (TAB-meristem) sprouting characterized by apical dominance (AD). Dormancy is shortened by treatments such as bromoethane (BE), which mimics the phenotype of dormancy release in cold storage by inducing early sprouting of several buds simultaneously. We studied the mechanisms governing TAB-meristem dominance release. TAB-meristem decapitation resulted in the development of increasing numbers of axillary buds with time in storage, suggesting the need for autonomous dormancy release of each bud prior to control by the apical bud. Hallmarks of programmed cell death (PCD) were identified in the TAB-meristems during normal growth, and these were more extensive when AD was lost following either extended cold storage or BE treatment. Hallmarks included DNA fragmentation, induced gene expression of vacuolar processing enzyme1 (VPE1), and elevated VPE activity. VPE1 protein was semipurified from BE-treated apical buds, and its endogenous activity was fully inhibited by a cysteinyl aspartate-specific protease-1-specific inhibitor N-Acetyl-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO). Transmission electron microscopy further revealed PCD-related structural alterations in the TAB-meristem of BE-treated tubers: a knob-like body in the vacuole, development of cytoplasmic vesicles, and budding-like nuclear segmentations. Treatment of tubers with BE and then VPE inhibitor induced faster growth and recovered AD in detached and nondetached apical buds, respectively. We hypothesize that PCD occurrence is associated with the weakening of tuber AD, allowing early sprouting of mature lateral buds.

Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall thickening after cessation of phellogen cell division.

Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition.

Snakin-1 (SN1) is an antimicrobial cysteine-rich peptide isolated from potato (Solanum tuberosum) that was classified as a member of the Snakin/Gibberellic Acid Stimulated in Arabidopsis protein family. In this work, a transgenic approach was used to study the role of SN1 in planta. Even when overexpressing SN1, potato lines did not show remarkable morphological differences from the wild type; SN1 silencing resulted in reduced height, which was accompanied by an overall reduction in leaf size and severe alterations of leaf shape. Analysis of the adaxial epidermis of mature leaves revealed that silenced lines had 70% to 90% increases in mean cell size with respect to wild-type leaves. Consequently, the number of epidermal cells was significantly reduced in these lines. Confocal microscopy analysis after agroinfiltration of Nicotiana benthamiana leaves showed that SN1-green fluorescent protein fusion protein was localized in plasma membrane, and bimolecular fluorescence complementation assays revealed that SN1 self-interacted in vivo. We further focused our study on leaf metabolism by applying a combination of gas chromatography coupled to mass spectrometry, Fourier transform infrared spectroscopy, and spectrophotometric techniques. These targeted analyses allowed a detailed examination of the changes occurring in 46 intermediate compounds from primary metabolic pathways and in seven cell wall constituents. We demonstrated that SN1 silencing affects cell division, leaf primary metabolism, and cell wall composition in potato plants, suggesting that SN1 has additional roles in growth and development beyond its previously assigned role in plant defense.

Hybrid proline-rich proteins: novel players in plant cell elongation?

BACKGROUND AND AIMS: Hybrid proline-rich proteins (HyPRPs) represent a large family of putative cell-wall proteins characterized by the presence of a variable N-terminal domain and a conserved C-terminal domain that is related to non-specific lipid transfer proteins. The function of HyPRPs remains unclear, but their widespread occurrence and abundant expression patterns indicate that they may be involved in a basic cellular process. METHODS: To elucidate the cellular function of HyPRPs, we modulated the expression of three HyPRP genes in tobacco (Nicotiana tabacum) BY-2 cell lines and in potato (Solanum tuberosum) plants. KEY RESULTS: In BY-2 lines, over-expression of the three HyPRP genes with different types of N-terminal domains resulted in similar phenotypic changes, namely increased cell elongation, both in suspension culture and on solid media where the over-expression resulted in enhanced calli size. The over-expressing cells showed increased plasmolysis in a hypertonic mannitol solution and accelerated rate of protoplast release, suggesting loosening of the cell walls. In contrast to BY-2 lines, no phenotypic changes were observed in potato plants over-expressing the same or analogous HyPRP genes, presumably due to more complex compensatory mechanisms in planta. CONCLUSIONS: Based on the results from BY-2 lines, we propose that HyPRPs, more specifically their C-terminal domains, represent a novel group of proteins involved in cell expansion.