Native Periderm Research Articles

An ARF1-binding factor triggering programmed cell death and periderm development in pear russet fruit skin.

Plants have a cuticular membrane (CM) and periderm membrane (PM), which act as barriers to terrestrial stresses. The CM covers primary organs with a continuous hydrophobic layer of waxes embedded in cutin, while the PM includes suberized cells stacked externally to the secondary tissues. The formation of native periderm is regulated by a postembryonic meristem phellogen that produces suberized phellem (cork) outwardly. However, the mechanism controlling phellogen differentiation to phellem remains to be clarified. Here, map-based cloning in a pear F1 population with segregation for periderm development in fruit skin facilitated the identification of an aspartic acid repeat deletion in Pyrus Periderm Programmed Cell Death 1.1 (PyPPCD1.1) that triggers phellogen activity for cork formation in russet fruit skin of pear. PyPPCD1.1 showed preferential expression in pear fruit skin, and the encoded protein shares a structural similarity to that of the viral capsid proteins. Aspartic acid deletion in PyPPCD1.1 weakened its nuclear localization but increased its accumulation in the chloroplast. The products of both PyPPCD1.1 and its recessive allele directly interact with ADP-ribosylation factor 1 (ARF1). PyPPCD1.1 triggered programmed cell death in an ARF1-dependent manner. Thus, this study identified the switch gene for programmed cell death and periderm development and provided a new molecular regulatory mechanism underlying the development of this trait.

“Periderm Disorder Syndrome”: a New Name for the Syndrome Formerly Referred to as Pink Eye

The “periderm disorder syndrome” (PDS), colloquially referred to previously as “pink eye”, is a physiological disorder caused by the death of the meristematically active layer of periderm cells (phellogen) and subsequent degeneration of the associated native periderm. This disorder occurs at a time when phellogen cell division is essential in the production of new periderm to cover and protect the expanding tuber surface and replace sloughed cells during later stages of rapid tuber growth. The characteristic degeneration and loss of periderm integrity, including the barrier provided by the suberized phellem, results in the aberrant induction of internal suberization. These aberrations often include accumulations of suberin polyphenolics (SPP) on neighboring cortical parenchyma cell walls as part of an erratic regenerative response to protect the vulnerable underlying tissues. Autofluorescence of the excessive accumulation of SPPs in cortical tissues located beneath the dysfunctional, or absent, periderm of afflicted tubers is a durable characteristic of extreme PDS. Conversely, the pinkish coloration sometimes associated with the PDS often is not present, and if present it is frequently barely discernable and ephemeral. Therefore, the pinkish coloration is not a reliable diagnostic nor does it lend itself to an appropriate name. Rather the syndrome is characterized by degeneration of the periderm and induction of erratic regenerative responses (mainly SPP accumulations) that are diagnostic and indicative of PDS. As such, “Periderm Disorder Syndrome” is a more accurate and descriptive name for this erratic and costly problem.

Potato native and wound periderms are differently affected by down-regulation of FHT, a suberin feruloyl transferase

Potato native and wound healing periderms contain an external multilayered phellem tissue (potato skin) consisting of dead cells whose cell walls are impregnated with suberin polymers. The phellem provides physical and chemical barriers to tuber dehydration, heat transfer, and pathogenic infection. Previous RNAi-mediated gene silencing studies in native periderm have demonstrated a role for a feruloyl transferase (FHT) in suberin biosynthesis and revealed how its down-regulation affects both chemical composition and physiology. To complement these prior analyses and to investigate the impact of FHT deficiency in wound periderms, a bottom-up methodology has been used to analyze soluble tissue extracts and solid polymers concurrently. Multivariate statistical analysis of LC-MS and GC-MS data, augmented by solid-state NMR and thioacidolysis, yields two types of new insights: the chemical compounds responsible for contrasting metabolic profiles of native and wound periderms, and the impact of FHT deficiency in each of these plant tissues. In the current report, we confirm a role for FHT in developing wound periderm and highlight its distinctive features as compared to the corresponding native potato periderm.

Anthocyanin synthesis in native and wound periderms of potato.

Skin color of red potatoes is due to accumulation of anthocyanins in the tuber periderm, a protective tissue that replaces the epidermis at an early stage of tuber development. The periderm consists of external layers of suberized phellem cells making up the skin, and internal layers of parenchyma-like phelloderm cells. Red pigmentation is an important marketing factor for red-skinned potatoes. However, injuries to the tuber surface, which are common in the potato industry, result in the development of a wound periderm that is devoid of the characteristic red coloration. To study the reason for these differences in anthocyanin accumulation, the expression level of anthocyanin biosynthesis genes and regulators was monitored in native and wound periderm using microarray analysis and quantitative polymerase chain reaction. We found significantly higher expression of the anthocyanin pathway in the phelloderm cells compared with the skin and tuber-flesh samples. However, in wound periderm, the anthocyanin pathway was strongly downregulated relative to the native periderm. This was true for two developmental stages of the native periderm--'immature', when the skin is prone to skinning injuries, and 'mature', following skin set--suggesting that anthocyanin synthesis continues postharvest. Wound-induced expression of steroidal glycoalkaloid glycosyltransferases, suberin-related 3-ketoacyl-CoA synthase and actin indicated that downregulation of the anthocyanin-specific pathway does not reflect global repression of the wound-periderm transcriptome. Loss of pigmentation may result from reduced expression of the Myb-bHLH-WD40 anthocyanin regulatory complex--a possible candidate might be the bHLH transcription factor JAF13.

Molecular and cytological aspects of native periderm maturation in potato tubers

Mature native periderm that exhibits resistance to excoriation (RE) is the primary defense for potato tubers against abiotic and biotic challenges. However, little is known about the physiology of periderm maturation and associated gene expressions. In this study, periderm maturation events and associated gene expressions were determined in tubers of two diverse potato genotypes (NDTX4271-5R (ND) and Russet Burbank (RB); 2008 and 2009 crops) at four harvest maturities ranging from immature (non-senesced vines and low RE) to mature (senesced vines and high RE). Approximately 104d after planting, the fine balance of accumulation and loss of periderm phellem cell layers showed signs of subsiding, indicating cessation of cell division by the phellogen. Phellogen radial cell walls thickened as periderm matured throughout the harvests, increasing RE/skin-set. In both genotypes, the cell cycle gene cyclin-dependent kinase B (StCDKB) rapidly down-regulated after the second harvest coinciding with apparent cessation of cell division. Expression patterns of genes encoding epidermal growth factor binding protein (StEBP) and cyclin-dependent kinase regulatory subunit (StCKS1At) were less indicative of phellogen inactivation and periderm maturation. Genes encoding the structural cell wall proteins extensin (StExt1) for ND and extensin-like (StExtlk) for ND and RB remained up-regulated respectively by the second harvest, suggesting involvement with completion of phellem cell accumulation and on-set of periderm maturation. The expression of genes encoding pectin methyl esterase (StPME), StExt1 and a cell wall strengthening “tyrosine-and lysine-rich protein” (StTLRP) increased in phellogen cells from later harvests of ND tubers, but were down regulated in RB tubers; this suggests roles in phellem cell generation and completion of delayed cell wall development in non-meristematic phellogen cells of ND, a red skinned phenotype. StCDKB and StPrePME genes were rapidly down-regulated by the third harvest for both genotypes. Collectively, these results suggest that down-regulation of these genes coordinates with on-set of periderm maturation and skin-set progression.

Proteomic evaluation of wound‐healing processes in potato (Solanum tuberosum L.) tuber tissue

Proteins from potato (Solanum tuberosum L.) tuber slices, related to the wound-healing process, were separated by 2-DE and identified by an MS analysis in MS and MS/MS mode. Slicing triggered differentiation processes that lead to changes in metabolism, activation of defence and cell-wall reinforcement. Proteins related to storage, cell growth and division, cell structure, signal transduction, energy production, disease/defence mechanisms and secondary metabolism were detected. Image analysis of the 2-DE gels revealed a time-dependent change in the complexity of the polypeptide patterns. By microscopic observation the polyalyphatic domain of suberin was clearly visible by D4, indicating that a closing layer (primary suberisation) was formed by then. A PCA of the six sampling dates revealed two time phases, D0-D2 and D4-D8, with a border position between D2 and D4. Moreover, a PCA of differentially expressed proteins indicated the existence of a succession of proteomic events leading to wound-periderm reconstruction. Some late-expressed proteins (D6-D8), including a suberisation-associated anionic peroxidase, have also been identified in the native periderm. Despite this, protein patterns of D8 slices and native periderm were still different, suggesting that the processes of wound-periderm formation are extended in time and not fully equivalent. The information presented in this study gives clues for further work on wound healing-periderm formation processes.

Relationship Between Pink Eye Symptoms and Cell Damage in the Tuber Periderm and Cortex

Pink eye (PE) is a tuber disorder that leads to processing complications and bud-end rot in storage. Despite the significance of PE for the potato processing industry, limited progress has been made in understanding the physiological basis of this disorder. Although the internal autofluorescence that characterizes PE has been described, the external symptoms of PE have not been well characterized or correlated with internal cellular symptoms. It is of vital importance to determine the physiological and cytological effects of PE in order to understand how it is caused and to develop effective management strategies to prevent the disorder. External symptoms progressed from pinkish discoloration around tuber eyes to water-soaked and corky patch lesions. Internal symptoms progressed from erratic browning around cortical cell walls to necrotic zones and cells with dark, circular inclusions. These areas often became surrounded by an internal PE-related periderm, while the native periderm often became compromised or lost. Vital staining with fluorescein diacetate showed that cortical and periderm cells underneath the native phellem became non-viable in PE tissue. Immunolocalization of homogalacturonan and extensin epitopes indicated that these cell wall polymers were not altered in pink eye afflicted tissues. These results conclusively show that the PE disorder results in or is caused by cell death in tissue underneath the native phellem, but does not lead to either cell wall breakdown or extensin deposition which is often characteristic of pathogen activity.

The Canon of Potato Science: 43. Skin-set and Wound-healing/Suberization

What is it?The native periderm covering the surface of the potato tuber is composed of phellem,phellogen and phelloderm cell layers. Immature periderm is fragile and susceptible toexcoriation,i.e. skinning injury, at the time of harvest and handling into storage. As theperidermmatures,theskin(phellemcelllayeroftheperiderm)becomestightlyattachedto the underlying cells and thereby resistant to skinning injury. The idiom “skin-set” isusedtoindicatethatthe tuber has becomeresistant toskinninginjury. Some documentsrefertoskinningasfeatheringbecauseofthethinandlightnatureofthephellem afteritis detached from the tuber. Wound-related blemishes, defects, infections and otherquality problems resulting from skinning injury necessitate the development of newapproachesandtechnologiestohastenandenhanceskin-set.However,untilrecentlythedirection for development of new means to improve skin-set was haphazard andseverely hampered because the physiological bases for skin-set and associatedbiological processes were not known. These knowledge gaps were significant and ledto many incorrect assumptions and hypotheses concerning skin-set development andhow the problem should be addressed. For example, one popular and incorrecthypothesis was that skin/phellem thickening was responsible for skin-set development.Another incorrect hypothesis ascribed suberization as the source of skin-set develop-ment.Recentidentificationofthebroadphysiologicalprocessesinvolvedintheskin-setphase of periderm maturation not only refute these hypotheses, but also guide researchin the proper direction for development of approaches to enhance skin-set.The cell walls of the tuber phellem/skin of the native periderm possess a waxycomponent that protects against cell desiccation and a protective suberin biopolymerthat provides a barrier to pathogens and other intrusions. The suberin biopolymer iscomposed of a suberin poly(phenolic) (SPP) domain that protects against bacterialinfections,andasuberinpoly(aliphatic)(SPA)domainthatprotectsagainstinfectionby

Pink eye is an unusual periderm disorder characterized by aberrant suberization: A cytological analysis

Potato tuber pink eye (PE) is a disorder of unknown origin that results in significant postharvest quality deterioration and rot. Little is known about the physiology of PE, including the characteristic tissue autofluorescence that defines the PE syndrome. The objective of this research was to identify the source of PE-induced autofluorescence and PE-related susceptibility to infection. The suberized barrier of the native periderm and cellular characteristics of neighboring parenchyma tissues were investigated to determine their involvement in the PE disorder. The results create a new physiological model describing the disorder and addressing the enigma of PE. Characteristics of the PE model emerge from the following results: (1) the integrity of the suberized barrier of the native periderm was compromised or absent in some surface areas of PE tubers thereby implicating the breakdown of the native periderm and its associated suberin barrier with PE and the susceptibility of PE tubers to pathogen infection; (2) the PE complex was characterized by unusual suberin poly(phenolic) (SPP) accumulations in the cortical parenchyma followed by latent suberin poly(aliphatic) (SPA) accumulations that were generally insufficient to form a complete barrier that was competent to block infections by pathogenic bacteria and fungi; (3) the aberrant absence or compromised integrity of the suberin barrier, including associated waxes, resulted in erratic increased susceptibility to water vapor loss known to cause tuber shrinkage and flaccidity; (4) widespread accumulations of SPP on parenchyma cell walls were the durable source of autofluorescence commonly used to determine the presence of the disorder; (5) the erratic development of unusual internal phellogen and periderm layers that, if complete with SPA, blocked hyphal advancement; (6) combined, the data provide a plausible explanation for PE infection court and rot anomalies as they occur without ingress of a wound opening. Results also demonstrated that neutral red may be used as a sensitive fluorochrome to detect intact hydrophobic areas in hyphae. Collectively, the results provide compelling evidence that the PE disorder includes a physiological basis.

Wax and suberin development of native and wound periderm of potato (Solanum tuberosum L.) and its relation to peridermal transpiration.

Native and wound periderm was isolated enzymatically from potato (Solanum tuberosum L. cv. Desirée) tubers at different time intervals between 0 days up to 4 weeks after harvesting. Wound periderm formation was induced by carefully removing native periderm from freshly harvested tubers before storage. The chemical composition of lipids (waxes) obtained by chloroform extraction, as well as the monomeric composition of native and wound suberin polymer after transesterification by boron trifluoride/methanol, was analyzed using gas chromatography and mass spectrometry. Both types of periderm contained up to 20% extractable lipids. Besides linear long-chain aliphatic wax compounds, alkyl ferulates were detected as significant constituents. In wound periderm they amounted to more than 60% of the total extracts. Within 1 month of storage, suberin amounts in the polymer increased 2-fold in native periderm (180 microg cm(-2)), whereas in wound periderm about 75.0 microg cm(-2) suberin polymer was newly synthesized. Native potato tuber periderm developed a very efficient transport barrier for water with permeances decreasing from 6.4 x 10(-10) m s(-1) to 5.5 x 10(-11) m s(-1) within 1 month of storage. However, the water permeability of wound periderm was on average 100 times higher with permeances decreasing from 4.7 x 10(-8) m s(-1) after 3 days to only 5.4 x 10(-9) m s(-1) after 1 month of storage, although suberin and wax amounts in wound periderm amounted to about 60% of native periderm. From this result it must be concluded that the occurrence of suberin with wax depositions in cell walls does not necessarily allow us to conclude that these cell walls must be nearly perfect barriers to water transport. In addition to the occurrence of the lipophilic biopolymer suberin and associated waxes, the still unknown molecular arrangement and precisely localized deposition of suberin within the cell wall must contribute to the efficiency of suberin as a barrier to water transport.

Immunocytological comparison of native and wound periderm maturation in potato tuber

The maturation of potato (Solanum tuberosum L.) tuber native periderm and wound periderm, which develops to replace the native periderm when it is damaged, are agriculturally important processes that are poorly understood. While both types of periderm form from a phellogen layer that serves as a lateral meristem, there has been little research done on comparing the biochemical processes and steps involved in the maturation of the two types of periderm. Here, we use immunological techniques to compare some of the cell wall changes during wound and native periderm maturation. Consistent with our recent work on native periderm, we demonstrate that toluidine blue O is also useful for distinguishing between suberized and non-suberized cells in wound periderm. More importantly, we use the immunological probes JIM5 and JIM7 to show that there is no increase in either un-esterified or esterified homogalacturonan pectin epitopes in phellogen walls accompanying wound periderm maturation. In contrast, as we previously described, native periderm maturation and resistance to excoriation (skinning) is accompanied by an increase in relatively un-esterified and esterified homogalacturonan pectin epitopes in the walls of phellogen cells. These results demonstrate that the biochemical processes responsible for maturation and resistance to excoriation differ between native and wound periderm. This dissimilarity between wound and native periderm maturation demonstrates the potential limitations in applying the wound periderm model to research on native periderm.

Histological analysis of the maturation of native and wound periderm in potato (Solanum tuberosum L.) Tuber.

Maturation of potato (Solanum tuberosum L.) tuber native and wound periderm and development of resistance to periderm abrasion were investigated utilizing cytological and histochemical techniques. Both native and wound periderm consist of three different tissues: phellem, phellogen and phelloderm. It was previously determined that the phellogen walls of immature native periderm are thin and prone to fracture during harvest, leading to periderm abrasion (excoriation). Phellogen walls thicken and become less susceptible to fracture upon maturation of the periderm, leading to resistance to excoriation. We now demonstrate that phellogen cells of immature wound periderm also have thin radial walls and that wound periderm abrasion is due to fracture of these walls. Maturation of the wound periderm is also associated with an increase in the thickness of the phellogen radial walls. Histological analysis with ruthenium red and hydroxylamine-FeCI2, which stain unesterified and highly methyl-esterified pectins, respectively, indicates that the phellogen cell walls of native and wound periderm differ significantly regardless of the stage of maturity. Results obtained by staining with ruthenium red and hydroxylamine-FeCI2 imply that phellogen cell walls of immature native periderm contain methyl-esterified pectin, but are lacking in unesterified (acidic) pectins. Maturation of native periderm is accompanied by an apparent increase in unesterified pectins in the walls of phellogen cells, which may allow for the strengthening of phellogen cell walls via calcium pectate formation. Histological staining of the phellogen walls of wound periderm, on the other hand, implies that these walls are deficient in pectins. Moreover, maturation of wound periderm is not accompanied by an increase in unesterified pectins in these walls. Since peroxidase is known to catalyse the cross-linking of cell wall polymers, we stained native and wound periderm for the presence of peroxidase utilizing guaiacol as a substrate. Peroxidase staining was strong in the phellogen walls of both immature and mature native periderm and we could not detect any differences in staining between them. Peroxidase staining was weak in the phellogen walls of immature wound periderm and was not detectably different in mature wound periderm. Peroxidase data imply that there are distinct differences between native and wound periderm, though our data do not indicate that changes in peroxidase activity are involved in the development of resistance to periderm abrasion that occurs upon maturation of the periderm. However, we cannot rule out the involvement in this process of peroxidase isozymes that have low affinity for the substrates utilized here.

Techniques for detecting and measuring developmental and maturational changes in tuber native periderm

We developed a technique to quantitate native periderm permeability during tuber growth and maturation by porometrically determining vapor conductances. We also describe a simple, inexpensive means of determining the corresponding weight per unit area of suberized phellem cells (skin) in native periderm during tuber growth and maturation as a means of assessing the developmental status of native periderm. The porometric technique is very sensitive and rapid. Porometrically derived vapor conductances of mature native periderms were measured without difficulty, and native periderms were found to release ~0.005 moles of water vapor/meter2 native periderm surface/sec. into a dry test atmosphere. Immature native periderm released up to 28 times more water vapor than mature periderm even though the phellem cells were histochemically determined to possess both major components of suberin. We demonstrated use of the porometric technique by determining maturational changes in periderm permeability of growing/immature tubers and mature tubers at harvest. The technique revealed that the native periderm of Russet Burbank tubers was more permeable and had the potential to lose more water vapor, until becoming permeably mature, than Norchip or Norland tubers harvested on the same dates. The dry weight of the skin (phellem tissue) of native periderm (mg/ cm2) for all three cultivars increased during tuber growth, but plateaued to a relatively constant level, or slightly declined, while plants were still alive during the latter period of tuber growth. During this period, periderm vameable conductances were undergoing substantial declines. Comparison of skin dry weights of immature tubers indicated that stem end regions of tubers were developmentally ahead of equatorial and bud end regions; this difference decreased as the tubers matured at harvest time and was less apparent with some cultivars.