Chilling induces sugar and ABA accumulation that antagonistically signals for symplastic connection of dormant potato buds.

Endodormancy (ED) is a crucial stage in the life cycle of many perennial plants. ED release requires accumulating a certain amount of cold exposure, measured as chilling units. However, the mechanism governing the effect of chilling on ED duration is poorly understood. We used the potato tuber model to investigate the response to chilling as associated with ED release. We measured the accumulation of specific sugars during and after chilling, defined as sugar units. We discovered that ED duration correlated better with sugar units accumulation than chilling units. A logistic function was developed based on sugar units measurements to predict ED duration. Knockout or overexpression of the vacuolar invertase gene (StVInv) unexpectedly modified sugar units levels and extended or shortened ED, respectively. Silencing the energy sensor SNF1-related protein kinase 1, induced higher sugar units accumulation and shorter ED. Sugar units accumulation induced by chilling or transgenic lines reduced plasmodesmal (PD) closure in the dormant bud meristem. Chilling or knockout of abscisic acid (ABA) 8'-hydroxylase induced ABA accumulation, in parallel to sweetening, and antagonistically promoted PD closure. Our results suggest that chilling induce sugar units and ABA accumulation, resulting in antagonistic signals for symplastic connection of the dormant bud.

An alternative pathway to plant cold tolerance in the absence of vacuolar invertase activity.

To cope with cold stress, plants have developed antioxidation strategies combined with osmoprotection by sugars. In potato (Solanum tuberosum) tubers, which are swollen stems, exposure to cold stress induces starch degradation and sucrose synthesis. Vacuolar acid invertase (VInv) activity is a significant part of the cold-induced sweetening (CIS) response, by rapidly cleaving sucrose into hexoses and increasing osmoprotection. To discover alternative plant tissue pathways for coping with cold stress, we produced VInv-knockout lines in two cultivars. Genome editing of VInv in 'Désirée' and 'Brooke' was done using stable and transient expression of CRISPR/Cas9 components, respectively. After storage at 4°C, sugar analysis indicated that the knockout lines showed low levels of CIS and maintained low acid invertase activity in storage. Surprisingly, the tuber parenchyma of vinv lines exhibited significantly reduced lipid peroxidation and reduced H2 O2 levels. Furthermore, whole plants of vinv lines exposed to cold stress without irrigation showed normal vigor, in contrast to WT plants, which wilted. Transcriptome analysis of vinv lines revealed upregulation of an osmoprotectant pathway and ethylene-related genes during cold temperature exposure. Accordingly, higher expression of antioxidant-related genes was detected after exposure to short and long cold storage. Sugar measurements showed an elevation of an alternative pathway in the absence of VInv activity, raising the raffinose pathway with increasing levels of myo-inositol content as a cold tolerance response.

Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato.

MAIN CONCLUSION: An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts' transgene expression and protoplasts' regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

Sucrose promotes stem branching through cytokinin.

Shoot branching is an important aspect of plant architecture because it substantially affects plant biology and agricultural performance. Sugars play an important role in the induction of shoot branching in several species, including potato (Solanum tuberosum L.). However, the mechanism by which sugars affect shoot branching remains mostly unknown. In the present study, we addressed this question using sugar-mediated induction of bud outgrowth in potato stems under etiolated conditions. Our results indicate that sucrose feeding to detached stems promotes the accumulation of cytokinin (CK), as well as the expression of vacuolar invertase (VInv), an enzyme that contributes to sugar sink strength. These effects of sucrose were suppressed by CK synthesis and perception inhibitors, while CK supplied to detached stems induced bud outgrowth and VInv activity in the absence of sucrose. CK-induced bud outgrowth was suppressed in vinv mutants, which we generated by genome editing. Altogether, our results identify a branching-promoting module, and suggest that sugar-induced lateral bud outgrowth is in part promoted by the induction of CK-mediated VInv activity.

Vacuolar processing enzyme translocates to the vacuole through the autophagy pathway to induce programmed cell death.

The caspase-like vacuolar processing enzyme (VPE) is a key factor in programmed cell death (PCD) associated with plant stress responses. Growth medium lacking a carbon source and dark conditions caused punctate labeling of 35S::VPE1-GFP (StVPE1-GFP) in potato leaves. Under conditions of carbon starvation, VPE activity and PCD symptoms strongly increased in BY-2 cells, but to a much lesser extent in VPE-RNAi BY-2 cells. During extended exposure to carbon starvation, VPE expression and activity levels peaked, with a gradual increase in BY-2 cell death. Histological analysis of StVPE1-GFP in BY-2 cells showed that carbon starvation induces its translocation from the endoplasmic reticulum to the central vacuole through tonoplast engulfment. Exposure of BY-2 culture to the macroautophagy/autophagy inhibitor concanamycin A led to, along with an accumulation of autophagic bodies, accumulation of StVPE1-GFP in the cell vacuole. This accumulation did not occur in the presence of 3-methyladenine, an inhibitor of early-stage autophagy. BY-2 cells constitutively expressing RFP-StATG8IL, an autophagosome marker, showed colocalization with the StVPE1-GFP protein in the cytoplasm and vacuole. RNAi silencing of the core autophagy component ATG4 in BY-2 cells reduced VPE activity and cell death. These results are the first to suggest that VPE translocates to the cell vacuole through the autophagy pathway, leading to PCD.Abbreviations: ATG: autophagy related; CLP: caspase-like protease; HR: hypersensitive response; PCD: programmed cell death; St: Solanum tuberosum; VPE: vacuolar processing enzyme.

High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.

Differential response to heat stress in outer and inner onion bulb scales.

The formation of brown protective skin in onion bulbs can be induced by rapid post-harvest heat treatment. Onions that are peeled to different depths and are exposed to heat stress show that only the outer scales form the dry brown skin, whereas the inner scales maintain high water content and do not change color. Our study demonstrates that browning of the outer scale during heat treatment is due to an enzymatic process that is associated with high levels of oxidation components, such as peroxidase and quercetin glucoside. De novo transcriptome analysis revealed differential molecular responses of the outer and inner scales to heat stress. Genes involved in lipid metabolism, oxidation pathways, and cell-wall modification were highly expressed in the outer scale during heating. Defense response-related genes such as those encoding heat-shock proteins, antioxidative stress defense, or production of osmoprotectant metabolites were mostly induced in the inner scale in response to heat exposure. These transcriptomic data led to a conceptual model that suggests sequential processes for the development of browning and desiccation of the outer scale versus processes associated with defense response and heat tolerance in the inner scales.

Etiolated Stem Branching Is a Result of Systemic Signaling Associated with Sucrose Level.

The potato (Solanum tuberosum) tuber is a swollen stem. Sprouts growing from the tuber nodes represent loss of apical dominance and branching. Long cold storage induces loss of tuber apical dominance and results in secondary branching. Here, we show that a similar branching pattern can be induced by short heat treatment of the tubers. Detached sprouts were induced to branch by the heat treatment only when attached to a parenchyma cylinder. Grafting experiments showed that the scion branches only when grafted onto heat- or cold-treated tuber parenchyma, suggesting that the branching signal is transmitted systemically from the bud-base parenchyma to the grafted stem. Exogenous supply of sucrose (Suc), glucose, or fructose solution to detached sprouts induced branching in a dose-responsive manner, and an increase in Suc level was observed in tuber parenchyma upon branching induction, suggesting a role for elevated parenchyma sugars in the regulation of branching. However, sugar analysis of the apex and node after grafting showed no distinct differences in sugar levels between branching and nonbranching stems. Vacuolar invertase is a key enzyme in determining the level of Suc and its cleavage products, glucose and fructose, in potato parenchyma. Silencing of the vacuolar invertase-encoding gene led to increased tuber branching in combination with branching-inducing treatments. These results suggest that Suc in the parenchyma induces branching through signaling and not by excess mobilization from the parenchyma to the stem.

Emergence of Leuconostoc mesenteroides as a causative agent of oozing in carrots stored under non-ventilated conditions.

Long-term storage and transport of post-harvest carrots (Daucus carota L.) require a low-temperature, high-relative-humidity environment, usually with low ventilation. Following long-term storage, a slimy exudate (oozing) often appears on the carrots, leading to severe spoilage. We characterized the environmental conditions leading to these symptoms and identified the causative agent. Simulation of non-ventilated storage conditions revealed accumulation of CO2 (to 80%) and ethanol (to 1000 ppm); then, a transparent exudate appeared on the carrot surface which, upon ventilation, developed into tissue browning and soft rot. Peels from oozing carrots contained over 10-fold the total bacterial counts of healthy carrots. The total peel microbiome was determined by 16S rDNA sequencing. During oozing stage, the surface of carrots incubated in a CO2 -rich (98%) environment harboured a bacterial population dominated by Lactobacillales and Enterobacteriales, differing markedly from those incubated in air. Three prevalent bacterial isolates from the oozing carrots were identified as Pantoea agglomerans, Rahnella aquatilis and Leuconostoc mesenteroides. Inoculation of carrot discs with L. mesenteroides, but not the others, induced oozing under high CO2 , suggesting that this bacterium is responsible for oozing of stored carrots. These findings should enable development of approaches to preventing carrot spoilage during long-term storage.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

Stronger sink demand for metabolites supports dominance of the apical bud in etiolated growth.

The potato tuber is a swollen underground stem that can sprout under dark conditions. Sprouting initiates in the tuber apical bud (AP), while lateral buds (LTs) are repressed by apical dominance (AD). Under conditions of lost AD, removal of tuber LTs showed that they partially inhibit AP growth only at the AD stage. Detached buds were inhibited by exogenous application of naphthaleneacetic acid (NAA), whereas 6-benzyladenine (6-BA) and gibberellic acid (GA3) induced bud burst and elongation, respectively. NAA, applied after 6-BA or GA3, nullified the latters' growth-stimulating effect in both the AP and LTs. GA3 applied to the fifth-position LT was transported mainly to the tuber's AP. GA3 treatment also resulted in increased indole-3-acetic acid (IAA) concentration and cis-zeatin O-glucoside in the AP. In a tuber tissue strip that included two or three buds connected by the peripheral vascular system, treatment of a LT with GA3 affected only the AP side of the strip, suggesting that the AP is the strongest sink for GA3, which induces its etiolated elongation. Dipping etiolated sprouts in labeled GA3 showed specific accumulation of the signal in the AP. Transcriptome analysis of GA3's effect showed that genes related to the cell cycle, cell proliferation, and hormone transport are up-regulated in the AP as compared to the LT. Sink demand for metabolites is suggested to support AD in etiolated stem growth by inducing differential gene expression in the AP.

The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores.

Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection.

Cellular and Molecular Changes Associated with Onion Skin Formation Suggest Involvement of Programmed Cell Death.

Skin formation of onion (Allium cepa L.) bulb involves scale desiccation accompanied by scale senescence, resulting in cell death and tissue browning. Understanding the mechanism of skin formation is essential to improving onion skin and bulb qualities. Although onion skin plays a crucial role in postharvest onion storage and shelf life, its formation is poorly understood. We investigated the mode of cell death in the outermost scales that are destined to form the onion skin. Surprisingly, fluorescein diacetate staining and scanning electron microscopy indicated that the outer scale desiccates from the inside out. This striking observation suggests that cell death in the outer scales, during skin formation, is an internal and organized process that does not derive only from air desiccation. DNA fragmentation, a known hallmark of programmed cell death (PCD), was revealed in the outer scales and gradually decreased toward the inner scales of the bulb. Transmission electron microscopy further revealed PCD-related structural alterations in the outer scales which were absent from the inner scales. De novo transcriptome assembly for three different scales: 1st (outer), 5th (intermediate) and 8th (inner) fleshy scales identified 2,542 differentially expressed genes among them. GO enrichment for cluster analysis revealed increasing metabolic processes in the outer senescent scale related to defense response, PCD processes, carbohydrate metabolism and flavonoid biosynthesis, whereas increased metabolism and developmental growth processes were identified in the inner scales. High expression levels of PCD-related genes were identified in the outer scale compared to the inner ones, highlighting the involvement of PCD in outer-skin development. These findings suggest that a program to form the dry protective skin exists and functions only in the outer scales of onion.

Storage temperature controls the timing of garlic bulb formation via shoot apical meristem termination.

MAIN CONCLUSION: Timing of bulb formation and floral stem induction in garlic is controlled by preplanting storage temperature and shoot apical meristem termination, probably via FLOWERING LOCUS T (FT) genes. Garlic is planted in the winter, undergoes a vegetative stage, then forms bulbs in response to increasing temperature and lengthening photoperiod. Herein, the storage conditions for propagation bulbs are shown to potentially affect future vegetative-stage length and timing of bulb formation. Storage temperatures of 2 or 33 °C inhibited internal bud growth. Levels of endogenous abscisic acid (ABA) and its inactive isomer trans-ABA were significantly higher in the internal bud of cloves stored at 33 vs. 2 °C, and exogenous ABA treatment before planting confirmed its inhibitory effect on foliage leaf development. Bulb formation started 30 and 60 days after planting of cloves stored at 2 and 33 °C, respectively. Warm storage temperature induced the formation of multiple leaves and cloves after planting. Plants from cloves stored at warm temperature developed a floral stem, whereas those from cold storage did not. Allium sativum FLOWERING LOCUS T1 (AsFT1) was upregulated 2.5- and 4.5-fold in the internal bud and storage leaf, respectively, after 90 and 150 days of cold vs. warm storage. Expression of AsFT4, expected to be antagonist to AsFT1, was 2- to 3-fold lower in the internal bud from cold storage. Expression of AsFT2, associated with floral termination, was 2- to 3- and 10- to 12-fold higher for cold vs. warm storage temperatures, in the internal bud and storage leaf, respectively. Early bulb formation, induced by cold storage, is suggested to inhibit normal foliage leaf development and transition of the shoot apical meristem to reproductive meristem, through regulation of FT genes.

In planta anthocyanin degradation by a vacuolar class III peroxidase in Brunfelsia calycina flowers.

In contrast to detailed knowledge regarding the biosynthesis of anthocyanins, the largest group of plant pigments, little is known about their in planta degradation. It has been suggested that anthocyanin degradation is enzymatically controlled and induced when beneficial to the plant. Here we investigated the enzymatic process in Brunfelsia calycina flowers, as they changed color from purple to white. We characterized the enzymatic process by which B. calycina protein extracts degrade anthocyanins. A candidate peroxidase was partially purified and characterized and its intracellular localization was determined. The transcript sequence of this peroxidase was fully identified. A basic peroxidase, BcPrx01, is responsible for the in planta degradation of anthocyanins in B. calycina flowers. BcPrx01 has the ability to degrade complex anthocyanins, it co-localizes with these pigments in the vacuoles of petals, and both the mRNA and protein levels of BcPrx01 are greatly induced parallel to the degradation of anthocyanins. Both isoelectric focusing (IEF) gel analysis and 3D structure prediction indicated that BcPrx01 is cationic. Identification of BcPrx01 is a significant breakthrough both in the understanding of anthocyanin catabolism in plants and in the field of peroxidases, where such a consistent relationship between expression levels, in planta subcellular localization and activity has seldom been demonstrated.

Induction of Rhizopus oryzae germination under starvation using host metabolites increases spore susceptibility to heat stress.

Sweetpotato is a nutritional source worldwide. Soft rot caused by Rhizopus spp. is a major limiting factor in the storage of produce, rendering it potentially unsafe for human consumption. In this study, Rhizopus oryzae was used to develop a concept of postharvest disease control by weakening the pathogen through induction of spore germination under starvation conditions. We isolated the sweetpotato active fractions (SPAFs) that induce spore germination and used them at a low dose to enhance spore weakening caused by starvation. Germination in SPAF at 1 mg/ml weakened the pathogen spores by delaying their ability to form colonies on rich media and by increasing their sensitivity to heat stress. The weakening effect was also supported by reduced metabolic activity, as detected by Alarmar Blue fluorescent dye assays. Spores incubated with SPAF at 1 mg/ml showed DNA fragmentation in some of their nuclei, as observed by TUNEL assay. In addition, these spores exhibited changes in ultrastructural morphology (i.e., shrinkage of germ tubes, nucleus deformation, and vacuole formation) which are hallmarks of programmed cell death. We suggest that induction of spore germination under starvation conditions increases their susceptibility to stress and, therefore, might be considered a new strategy for pathogen control.

Vicilin and the basic subunit of legumin are putative chickpea allergens.

IgE-mediated reactions to food allergens constitute a major health problem in industrialized countries. Chickpea is consumed in Mediterranean countries, and reportedly associated with IgE-mediated hypersensitivity reactions. However, the nature of allergic reactions to chickpea has not been characterized. A serum pool from paediatric patients allergic to chickpeas was used to detect IgE-binding proteins from chickpea seeds by immunoassay and immunoblot inhibition assay. Protein samples enriched in chickpea legumin and vicilin were obtained by anion exchange chromatography, and were identified by mass spectrometric analysis. IgE-immunoassays of globulin fractions from chickpeas revealed that vicilin (50 kDa) and the basic subunit of legumin (20 kDa) were bound by IgE from patient sera. Pea and lentil protein extracts strongly inhibited the IgE binding to chickpea globulin. We speculate that vicilin and the basic subunit of legumin are major chickpea allergens. Also, the globulin fraction of chickpea likely cross-reacts with the allergenic proteins of pea and lentil.

Can loss of apical dominance in potato tuber serve as a marker of physiological age?

The potato tuber constitutes a model system for the study of dormancy release and sprouting, suggested to be regulated by endogenous plant hormones and their balance inside the tuber. During dormancy, potato tubers cannot be induced to sprout without some form of stress or exogenous hormone treatment. When dormancy is released, sprouting of the apical bud may be inhibited by sprout control agents or cold temperature. Dominance of the growing apical bud over other lateral buds decreases during storage and is one of the earliest morphophysiological indicators of the tuber's physiological age. Three main types of loss of apical dominance (AD) affect sprouting shape. Hallmarks of programmed cell death (PCD) have been identified in the tuber apical bud meristem (TAB-meristem) during normal growth, and are more extensive when AD is lost following extended cold storage or chemical stress. Nevertheless, the role of hormonal regulation in TAB-meristem PCD remains unclear.

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.

Postharvest dark skin spots in potato tubers are an oversuberization response to Rhizoctonia solani infection.

Israeli farmers export 250,000 tons of potato tubers annually, ≈40,000 tons of which are harvested early, before skin set. In recent years, there has been an increase in the occurrence of dark skin spots on early-harvested potato tubers ('Nicola') packed in large bags containing peat to retain moisture. The irregular necrotic spots form during storage and overseas transport. Characterization of the conditions required for symptom development indicated that bag temperature after packing is 11 to 13°C and it reaches the target temperature (8°C) only 25 days postharvest. This slow decrease in temperature may promote the establishment of pathogen infection. Isolates from typical lesions were identified as Rhizoctonia spp., and Koch's postulates were completed with 25 isolates by artificial inoculation performed at 13 to 14°C. Phylogenetic analysis, using the internal transcribed spacer sequences (ITS1 and ITS2) of rDNA genes, assigned three isolates to anastomosis group 3 of Rhizoctonia solani. Inoculation of wounded tubers with mycelium of these R. solani isolates resulted in an oversuberization response in the infected area. With isolate Rh17 of R. solani, expression of the suberin biosynthesis-related genes StKCS6 and CYP86A33 increased 6.8- and 3.4-fold, respectively, 24 h postinoculation, followed by a 2.9-fold increase in POP_A, a gene associated with wound-induced suberization, expression 48 h postinoculation, compared with the noninoculated tubers. We suggest that postharvest dark spot disease is an oversuberization response to R. solani of AG-3 infection that occurs prior to tuber skin set.