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JXB Advance Access originally published online on September 14, 2009
Journal of Experimental Botany 2009 60(15):4411-4421; doi:10.1093/jxb/erp281
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Transcriptomic profiling of heat-stress response in potato periderm

Idit Ginzberg1,*, Gilli Barel1, Ron Ophir1, Enosh Tzin1, Zaccharia Tanami1, Thippeswamy Muddarangappa1, Walter de Jong2 and Edna Fogelman1

1Institute of Plant Sciences, ARO, Volcani Center, PO Box 6, Bet Dagan 50250, Israel
2Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 14853-1901, USA

* To whom correspondence should be addressed: E-mail: iditgin{at}volcani.agri.gov.il

Received 14 June 2009; Revised 23 August 2009 Accepted 25 August 2009


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
 Supplementary data
 References
 
Potato (Solanum tuberosum L.) periderm is composed of the meristematic phellogen that gives rise to an external layer of suberized phellem cells (the skin) and the internal parenchyma-like phelloderm. The continuous addition of new skin layers and the sloughing of old surface layers during tuber maturation results in smooth, shiny skin. However, smooth-skin varieties frequently develop unsightly russeting in response to high soil temperatures. Microscopic observation of microtubers exposed to high temperatures (37°C) suggested heat-enhanced development and accumulation of suberized skin-cell layers. To identify the genes involved in the periderm response to heat stress, skin and phelloderm samples collected separately from immature tubers exposed to high soil temperatures (33°C) and controls were subjected to transcriptome profiling using a potato cDNA array. As expected, the major functional group that was differentially expressed in both skin and phelloderm consisted of stress-related genes; however, while the major up-regulated phelloderm genes coded for heat-shock proteins, many of the skin's most up-regulated sequences were similar to genes involved in the development of protective/symbiotic membranes during plant–microbe interactions. The primary activities regulated by differentially expressed peridermal transcription factors were response to stress (33%) and cell proliferation and differentiation (28%), possibly reflecting the major processes occurring in the heat-treated periderm and implying the integrated activity of the stress response and tissue development. Accumulating data suggest that the periderm, a defensive tissue, responds to heat stress by enhancing the production and accumulation of periderm/skin layers to create a thick protective cover. Skin russeting may be an indirect outcome; upon continued expansion of the tuber, the inflexible skin cracks while new layers are produced below it, resulting in a rough skin texture.

Key words: Heat stress, microtubers, phellem, phelloderm, phellogen, skin russeting, Solanum tuberosum


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Concluding remarks
 Supplementary data
 References
 
The original epidermal cell layer of the tuber is short-lived, replaced early in development with periderm tissue that is made up of three cell types: phellem, phellogen, and phelloderm (Reeve et al., 1969). The phellem (or cork) forms a series of layers at the outermost level of the periderm, and is derived from the meristematic phellogen layer (or cork cambium) below it. Cork is composed of suberin macromolecules that consist of aromatic and aliphatic polyester domains cross-linked by glycerol and is localized between the primary wall and the plasmalemma (Kolattukudy, 1984). Hence, as phellem cells become suberized and die, they form a protective layer that is designated as ‘skin’. The phelloderm cells form the innermost layer of the periderm, and are similarly derived from the phellogen layer, which is located directly above them. New skin layers are continuously added by cell divisions during tuber maturation. In immature periderm, the actively dividing phellogen is labile and prone to fracture, allowing separation of the suberized phellem/skin from the underlying phelloderm and tuber flesh (Lulai and Freeman, 2001; Sabba and Lulai, 2002). In the present work, this characteristic was used to obtain homogeneous skin tissue.

Both periderm components, the skin and the phelloderm, contribute to its protective characteristics. The corky skin cells are filled with air and therefore provide thermal insulation, the suberized walls prevent invasion by micro-organisms (mechanically and chemically), and wax deposits that are embedded within the suberin material prevent desiccation of internal tissues (Kolattukudy, 1977, 1984). Immature skin is also enriched with proteins involved in defence responses to biotic and abiotic stresses (Barel and Ginzberg, 2008). Likewise, the parenchyma-like phelloderm is enriched with defensive secondary metabolites (I. Ginzberg, unpublished data; Krits et al., 2007).

The periderm remains thin during tuber development via the loss of superficial cork cells, rendering the skin smooth and shiny; however, high soil temperatures induce severe russeting (Fig. 1). Limited comparisons to date have shown that tubers with genetically inherited russeted skin have a thicker layer of phellem than smooth-skinned potatoes (Yamaguchi et al., 1964; Okazawa and Iriuda, 1980). This observation led to the suggestion that russeted skin results when phellem cells adhere so tightly to each other that they are not sloughed off during tuber development (Yamaguchi et al., 1964; Okazawa and Iriuda, 1980). The basis for increased cohesion might be increased suberization (Yamaguchi et al., 1964) or increased levels of pectin and hemicellulose (Okazawa and Iriuda, 1980).


Figure 1
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  		Fig. 1. Heat stress russeting. Cross-sections (A, C) of Desirée tubers (B, D) collected from a commercial field in Israel at the end of the growth season (beginning of July). Samples were stained with Safranin O/Fast green to view tissue morphology.

 
A study on the inheritance of russeting suggests complementary action by three independently segregating genes, A, B, and C, where a dominant allele at each locus is required for any degree of skin russeting (de Jong, 1981). Nevertheless, the roughness of Russet Burbank skin is also dependent on growth temperatures (Yamaguchi et al., 1964), suggesting that common genes for heat-induced russeting and ‘conventional’ russeting might be involved.

A histological approach was used to characterize the morphology of heat-stressed periderm and a potato cDNA array was used to identify genes that may play a role in heat-induced russeting. Differentially expressed clones were sorted into functional categories and, as expected, the main category was related to the biotic and abiotic stress responses. Nevertheless, 30% of differentially expressed transcription factors and signalling genes appeared to be involved in the regulation of cell proliferation and differentiation, supporting the microscopic observation of the enhanced accumulation of suberized skin layers.

The data presented here may also contribute to understanding the effect of high temperature on periderm development in other plant systems, such as cork in woody species, root phellem, and wound-healing.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Concluding remarks
 Supplementary data
 References
 
Plant material
Microtubers of potato (Solanum tuberosum L.) cultivar Desirée were induced according to standard protocol (Donnelly et al., 2003). Single-node stem cuttings were placed on Nitsch medium (Sigma Chemicals, Rehovot, Israel) (pH 5.8) containing 8% (w/v) sucrose, 5 mg l–1 kinetin (Sigma), 2 mg l–1 ancimidol (Sigma) and 0.8% (w/v) agar (Sigma) in a Petri dish. The cuttings were incubated in the dark at 24°C, and, after 7 d, 1–2 mm microtubers could be observed. At this stage, periderm tissue with suberized phellem (skin) starts replacing the epidermis; the periderm continues to develop throughout microtuber expansion. To monitor periderm development, 7 d after microtuber induction, stem cuttings with developing microtubers were transferred to 37°C or 15°C, or maintained at 24°C as a control. Microtubers were collected 2, 7, and 21 d later for histological observation.

For the transcript-profiling study, Desirée plants were grown in pots (20 l) filled with perlite in a greenhouse under natural winter conditions (November to January, average temperature range of 10–18°C). Tubers were harvested at the end of the eighth week post-sprout emergence; at this developmental stage, the skin (12–14 cell layers) is easily peeled by hand from the tuber flesh exposing the phelloderm layers, which were collected using a blade. The resulting phelloderm sample was about seven cell layers thick (equal to 200 µm, measured under a light microscope; I Ginzberg, unpublished data); minor contamination with flesh parenchyma cells could not be ruled out.

For the exposure of tubers to heat stress, hot water (33–35°C) was circulated in tubes lining the internal side of the pots. The heat was applied for 1 week before tuber harvest. Plants were watered every day to prevent drying out of the perlite and induction of drought stress; heat-treated plants looked as healthy as control plants.

Skin and phelloderm samples from normally grown or heat-stressed tubers were snap-frozen in liquid nitrogen and stored at –80°C. For each sample, two biological replicates were prepared, each with tissues pooled from five plants grown at different locations in the greenhouse.

Tissue embedding and histological staining
Tissue samples were fixed in FAA (50% ethanol, 5% acetic acid, and 3.7% formaldehyde, by vol.), dehydrated in an ethanol/Histo-clear (Finkelman Chemicals, Petach-Tikva, Israel) series, and embedded in paraplast (Paraplast Plus, McCormick Scientific, St Louis, MO) according to standard methods (Ruzin, 1999). Tissue sections (15–20 µm) were stained with Safranin O/Fast green (Sigma) for examination of tissue morphology (Johansen, 1940). Sections were observed under a light microscope (Leica DMLB, Wetzlar, Germany) and images were displayed on a monitor through a CCD camera (Leica DC2000) using the Leica IM1000 program. The same samples were viewed under UV light to detect autofluorescence of suberized cell walls in the skin: the Leica DMLB microscope was configured for epifluorescent illumination using an HBO103W/2 mercury lamp, excitation filter BP 340–380, chromatic beam-splitter FT 400, and barrier filter LP 425.

Total RNA extraction and quantitative real-time PCR
RNA was isolated as described by Chang et al. (1993). After LiCl precipitation, total RNA was washed in 70% cold ethanol, vacuum-dried for 5 min, and washed again in a mixture consisting of equal parts of SSTE (1 M NaCl, 0.1 M TRIS pH 8, 0.5% w/v SDS, and 0.001 M EDTA) in diethylpyrocarbonate (DEPC)-treated double-distilled water and acidic phenol:chloroform:isoamyl alcohol solutions. After removal of the organic phase, the RNA was reprecipitated overnight in 100% ethanol at –20°C and centrifuged (4°C, 15 min, 18 000 g). The RNA pellet was washed again in 70% cold ethanol, dried on ice for 15 min, and redissolved in 30 µl of DEPC-treated double-distilled water. Total RNA was quantified using a NanoDrop® ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE). RNA was further purified by RNeasy Mini Kit (Qiagen, Hilden, Germany) using the On-Column DNase Digestion protocol.

Quantitative real-time PCR was performed using the EZ-First Strand cDNA Synthesis Kit for RT-PCR (Biological Industries, Beit Haemek, Israel) and ABsoluteTM Blue QPCR SYBR® Green ROX Mix (Thermo Scientific, Surrey, UK) according to the manufacturers’ protocols, with specific primers (Table 1). Each PCR was performed with three technical replicates.


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  		Table 1. List of primers used for quantitative real-time PCR

 
Microarrays
The TIGR Solanaceae Expression Profiling Service performed all of the microarray procedures, including two-colour cDNA labelling, hybridization, data quantification, and data normalization (protocols available at http://www.jcvi.org/potato/sol_ma_protocols.shtml). The data from the microarray experiments are available from the TIGR Solanaceae Gene Expression Database (http://www.jcvi.org/potato/sol_expression.shtml). The TIGR 10K EST Solanaceae microarray contained 11 412 verified cDNA clones spotted as randomized duplicates on the array (http://www.jcvi.org/potato/sol_ma_microarrays.shtml). Hybridizations were performed with four tissue samples: skin (S), heat-stressed skin (S[H]), phelloderm (Ph), and heat-stressed phelloderm (Ph[H]), each in two biological replicates, and each replicate was labelled with each of the dyes (once red and once green, i.e. two technical replicates for each biological replicate). Hybridizations were performed in a loop design as described in Yang and Speed (2002). One technical replicate was used to create the loop design in a clockwise direction and the other technical replicate duplicated the loop in a counter-clockwise direction (see Supplementary Fig. S1 at JXB online).

Data analysis
The eight RNA samples were hybridized to the TIGR Solanaceae array. R packages from the Bioconductor project (Gentleman et al., 2004) were used. Initially, per-tip-loss normalization was performed (Yang et al., 2002), followed by a statistical test for differentially expressed genes using the Linear Models for Microarray package (Smyth, 2004), which allows better variance estimation by calculating the moderated t statistic using an empirical Bayesian approach. The probes’ log signal ratios were ranked by their adjusted P-value (q-value), and selected for genes with significantly different expression (q-value <0.05). The correction for multiple comparisons was done using Benjamini and Hochberg's (1995) false discovery rate (FDR). Moreover, the internal variation of technical replicates was estimated and used for weighting the fitting of the biological replicates. This approach is equivalent to the randomized block previously described in Smyth et al. (2005).

Annotations of all the differentially expressed genes were verified by nucleotide alignment or searching the protein database with a translated sequence of the respective clone using NCBI BLAST. Data are updated to March 2009.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Concluding remarks
 Supplementary data
 References
 
Enhanced suberized skin development at high temperatures
Tissue culture of in vitro-generated microtubers was used as a model system to follow changes occurring in the skin upon exposure to high temperatures. Histological observation showed enhanced suberized skin development at 37°C, 2 d after transfer to high temperature (Fig. 2B). Autofluorescence of suberized cells could not be observed at this stage in control microtubers (Fig. 2H). Continued incubation at 37°C resulted in the development of multiple suberized skin layers with a condensed appearance (Fig. 2D, F), while their respective controls exhibited well-organized skin layers with the characteristic morphology of cell columns (Fig. 2J, L). The effect of temperature on periderm development was further demonstrated by incubating the developing microtubers at 15°C, where suberized skin development was delayed compared with the controls (Fig. 2N, P, R).


Figure 2
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  		Fig. 2. Temperature effect on suberized skin development. Seven-day-old microtubers were transferred to high (37°C, A–F), optimal (24°C, G–L) or low (15°C, M–R) growth temperatures. Microtubers were collected 2, 7, and 21 d later, embedded in paraplast, sectioned to 15 µm, dyed with Safranin O/Fast green, and inspected under the light (left photograph of each treatment) or UV (right photograph of each treatment) microscope to examine tissue morphology and autofluorescence of suberized cells, respectively. Bar=200 µm.

 
An increase in the number of skin/periderm cell layers in response to elevated temperatures has been reported previously for russeted cultivars of potato (Yamaguchi et al., 1964; Okazawa and Iriuda, 1980), as well as for sweet potato periderm (Villavicencio et al., 2007). The skin of Russet Burbank tubers showed increased roughness with increasing soil temperature (Yamaguchi et al., 1964). At cool temperatures the periderm (skin and phelloderm) consisted of 7–12 layers and was 100–140 µm thick with a smooth surface, while at 24–27°C its surface was cracked and russeted (patches of thick tissue surrounded by areas of thin and smoothed tissue) consisting of up to 28 layers of cells (270 µm thick) in the russeted areas. In addition, at the highest soil temperature, the inner layers of the suberized skin appeared to be collapsed and to contain more pectic substance than their low-temperature-grown counterparts. The authors suggested that the increase in periderm thickness is due to increased activity of the phellogen under high temperature. The existence of masses of cells in the russeted areas suggested strong heat-associated adherence between the suberized skin cells, which are therefore not easily sloughed off compared to conditions of low soil temperature (Yamaguchi et al., 1964). Okazawa and Iriuda (1980) suggested that russeting evolves from secondary tuber growth in response to high temperature: upon resumption of tuber expansion, the primary periderm is cracked but remains as patches that adhere to the new skin cells that form below (Fig. 1).

The data suggest that during periods of high temperature, periderm development is accelerated and layers of suberized skin cells accumulate, reducing the flexibility of the skin. As the tuber continues to expand, the outer layers of the skin are fissured and new skin cells are formed underneath by the active phellogen. Thus, the surface of the tuber loses its smooth and shiny appearance and become russeted.

The periderm is a protective tissue (Kolattukudy, 1977, 1984) that contains a wide array of plant defence components (Barel and Ginzberg, 2008). The enhanced activity of the meristematic phellogen could be part of its mechanism of confronting heat stress, by creating an insulating cover and preventing water loss from the tuber flesh.

Heat-induced differential expression of periderm transcriptome
The transcriptomes of native and heat-stressed periderm were analysed to identify factors involved in the above-described morphological changes. The periderm components of skin and phelloderm were separated and analysed separately using a potato cDNA array (TIGR Solanaceae array) to portray the characteristic response of each to high soil temperatures. Data analysis resulted in three lists: S[H] and Ph[H] (see Supplementary Tables S1 and S2 at JXB online) which contain genes whose expression was altered mainly in the heated skin or phelloderm, respectively, and SPh[H] (see Supplementary Table S3 at JXB online) which contains genes with similar expression patterns in both tissues. The clones from these lists were compared to a previous study in which the same cDNA arrays were used to identify genes with altered expression in potato seedlings that had been exposed to heat, cold, and salt stresses (Rensink et al., 2005). Clones that appeared in the present work and in that of Rensink et al. (2005) are marked with an asterisk in Supplementary Tables S1, S2 and S3 at JXB online, and those of unknown function were categorized as stress-related. Unmarked clones may represent activities that highlight the unique response of the periderm to high soil temperatures.

Clone annotations and their functional categories were determined based on alignment studies and a literature survey. About 14–18% of the clones in each tissue were of unknown function and 7–12% were classified as miscellaneous (Fig. 3). As expected, clones categorized as stress-related were the main functional groups in each tissue: 18.6% of the S[H] clones, 28.7% of the Ph[H] clones, and 20% of the shared clones in SPh[H]. Some clones that were classified to other functional categories may also play a role in plant defence (as discussed later).


Figure 3
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  		Fig. 3. Graphical representation of the percentage of differentially expressed genes that belong to a given functional group, in (A) heat-treated skin (S[H]) and (B) heat-treated phelloderm (Ph[H]), and (C) with similar expression patterns in both peridermal components (SPh[H]). Values were calculated per total number of differential clones in each tissue (given in brackets), and are given at the head of the bars; white and grey bars represent up- and down-regulated expression, respectively.

 
At first glance, the differentially expressed clones did not provide a comprehensive understanding of the processes that occur during heat stress; however, close examination of the ‘regulation of transcription’ category gave a clue as to the factors/processes that may maintain the proliferating activity of the periderm and alter its morphology.

For clarity, genes that are discussed in the following text are followed by a subscript indicating their tissue location (S, Ph or SPh), a ‘minus’ sign indicating down-regulation where relevant, and their ordinal number in the corresponding tables (see Supplementary Tables S1, S2 or S3 at JXB online). In addition, most of the genes named below are not potato genes per se, but rather are functionally-characterized genes from other species that share substantial sequence similarity to the corresponding cDNA clones on the microarray.

Tissue-related heat-induced differential expression of transcription factors and related activities
An examination of the transcription factors that are mainly altered in the skin following heat treatment (constituting 9.5% of all S[H] clones; Fig. 3) suggested the integrated regulation of tissue development and stress responses (Table 2). Thirty-five per cent of these transcription factors may regulate cell proliferation, orientation, and differentiation. For example, GRAS10/SCL8S39, GRAS25/SCL6S-165, and GeBP-likeS50 have been reported to be expressed in developing tissues. The two former transcription factors belong to a family of proteins involved in tissue patterning (Pysh et al., 1999) and the latter is expressed in vegetative meristematic tissues (Curaba et al., 2003). Additional transcription factors with altered expression were: AOBPS-258,-246, a negative regulator of cell growth whose mRNA exhibits reduced or no expression in growing tissues (Kisu et al., 1998), CAM7S-205 which regulates light-dependent growth developmental patterns (Kushwaha et al., 2008), and a KH domain-containing proteinS-132 whose orthologues are involved in cell differentiation and apoptosis induction (Lorkovic and Barta, 2002). In addition, SHRS-126 (Helariutta et al., 2000) and LOBS-155 (Iwakawa et al., 2007) are required for asymmetric cell division in meristems.


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  		Table 2. List of transcription factors with significant (P-value <0.05) differential expression in heat-stressed skin compared to heat-stressed phelloderm

 
Differentially-expressed stress-related transcription factors (38.5%) detected include the BTB/TAZ domain protein-5S11 (Du and Poovaiah, 2004) and TGA1a homologueS45 (Niggeweg et al., 2000), which bind to functional elements of plant promoters to direct a systemic response upon pathogen attack; both are also activated by salicylic acid. MYB150-likeS69, and zinc-finger DNA-binding proteinS76 (Rensink et al., 2005) respond to abiotic stresses while ELF3S59 may be involved in compensation of the central circadian clock in the face of temperature stress (Boxall et al., 2005). Interestingly, three of the down-regulated transcription factors may regulate response to drought: HB-7S-269 (Soderman et al., 1996), CPHB-5 orthologueS-216 (Deng et al., 2002), and AREB-like proteinS-164. Other stress-related transcription factors observed were B-box zinc fingerS-218, a putative salt-tolerance protein, and AG motif-binding protein-1S-128, a positive regulator of wounding and elicitor response (Sugimoto et al., 2003).

Similarly, the most highly up-regulated S[H] genes suggest integrated activities of tissue development and stress response (see Supplementary Table S1 at JXB online). These include plant–microbe interaction-related sequences such as the root-knot nematode giant cell transcriptS5 and miraculinS10 (Brenner et al., 1998), as well as early nodulin-93S2 expressed in the primary nodule meristem (Kouchi and Hata, 1993). These sequences play a role in the development of protective/symbiotic cells/membranes while their S[H] orthologues may play a role in heat-related development of tuber skin. This hypothesis is further supported by the high expression of ACC oxidaseS1, which is involved in stress responses (Abeles et al., 1992; Zanetti et al., 2002) but was also shown to be dramatically induced prior to wound-periderm development (Gerchikov et al., 2008), as well as the high expression of genetic-tumour-related proteinS6 (Fujita et al., 1994). Parallel to the induction of these genes, there was down-regulation of others involved in programmed cell death and senescence, which may result in maintaining the periderm in a ‘juvenile’ proliferating state. Down-regulated genes include BAG-domain-containing proteinS-265 (Doukhanina et al., 2006), pirin-like proteinS-263 (Orzaez et al., 2001), metallothionein-like protein type-2S-231 (Harvey et al., 2008), bifunctional nuclease BFN1S-203 (Farage-Barhom et al., 2008), Rac-likeS-197 protein (Hwang et al., 2008), and Sen-1 like proteinS-178. The combined effect of all these S[H] genes may result in enhanced proliferation of skin layers in response to heat stress (Fig. 2).

Transcription regulators that exhibited altered expression in the phelloderm-enriched layers (constituting 5.4% of all the Ph[H] clones, Fig. 3) appear also to regulate both cell proliferation and stress response (Table 3). Examples are MSI2Ph43, which codes for a retinoblastoma-associated protein involved in the regulation of G1 progression of the cell cycle and may be related to heat-shock responsiveness (Ach et al., 1997), and stress-related WRKYPh18,56 family members coding for pathogen- and salicylic acid-induced transcription factors (Chen and Chen, 2000).


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  		Table 3. List of transcription factors with significant (P-value <0.05) differential expression in heat-stressed phelloderm compared to heat-stressed skin

 
Note that, in contrast to the S[H] clones, the major up-regulated Ph[H] clones (fold change [FC] >1.8) were heat-shock proteins, while the most significant down-regulated Ph[H] clones (FC <–1.8) were protease inhibitors (see Supplementary Table S2 at JXB online).

Analysis of sequences with similar expression patterns in both S[H] and Ph[H] tissues that are involved in the regulation of transcription (8.7% of total clones in SPh[H], Fig. 3) resulted in functions similar to those already described (Table 4): regulation of tissue differentiation (28.2%), and stress responses (32.6%), were the main activities. An example of a transcription factor that may be involved with periderm development is TT1-likeSPh2, which is also required for proanthocyanidin biosynthesis by channelling anthocyanin precursors to the production of condensed tannins (Sagasser et al., 2002). Its high expression in the heat-treated periderm may explain the pigment loss in red-skin potatoes when tubers are exposed to high soil temperatures.


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  		Table 4. List of transcription factors with significant (P value <0.05) differential expression in heat-stressed skin and phelloderm compared to control

 
Half of the stress-related transcription factors were up-regulated clones of WRKYSPh12,13,15,48,67,68,249 (Chen and Chen, 2000; Wu et al., 2007); these family members differ from the Ph[H] up-regulated WRKYPh18,56 genes. Additional SPh[H] transcription factors are listed in Table 4.

The functions of genes assigned to signal transduction in heat-treated skin and phelloderm were also in accordance with developmental activity of the periderm combined with the stress response. A high number of S[H] clones were implicated in signalling (8.3%; Fig. 3). Most of these participate in stress-related pathways (65%) (see Supplementary Table S1 at JXB online), while down-regulation of two clones, Rac-likeS-197 (Hwang et al., 2008) and pirinS-263 (Orzaez et al., 2001), probably enable the continuation and orientation of cell proliferation. In Ph[H], 3.5% of the clones were assigned to signalling: 56% of these are implicated in stress signalling and 33% in cell division and polarity (see Supplementary Table S2 at JXB online). Most of the SPh[H] signalling clones (64%) have unknown activities, while 27% may support periderm development (see Supplementary Table S3 at JXB online).

To validate the microarray analysis, the expression level of eight randomly selected SPh[H]-transcription factor clones (five up-regulated and three down-regulated) was analysed by quantitative real-time PCR in heat-treated and control skin and phelloderm samples (Fig. 4). Values were normalized to the level of the NAC/UBA-like (TA25576) reference gene (see Supplementary Table S4 at JXB online; Barel and Ginzberg, 2008) in each sample, and then relative to their corresponding levels in the skin control sample. The expression patterns obtained were in accordance with the SPh[H] microarray analysis: clones were up- or down-regulated in both S[H] and Ph[H] samples, and the relative transcript level was proportional to the microarray FC value (Fig. 4).


Figure 4
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  		Fig. 4. Differential expression of transcription factors in heated and control periderm components as verified by quantitative real-time PCR. Transcription factors with similar expression patterns in both skin and phelloderm were selected at random from the SPh[H] list (Table 4) and their level was quantified by real-time PCR in each of the components separately. RNA samples consisted of two biological repeats, each collected from five independent plants. Expression levels were normalized to the level of NAC/UBA-like (TA25576) reference gene in each sample. Bars represent expression levels in the phelloderm (Ph), heated skin (S[H]), and heated phelloderm (Ph[H]), relative to the levels in the skin (S) sample (S values equal to 1). Gene names are followed by a subscript indicating their ordinal number in Supplementary Table S3 at JXB online, and fold change values obtained in the microarray analysis in brackets: a minus sign indicates down-regulation.

 

   Concluding remarks
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
Supplementary data
 References
 
Examination of the biological functions of the heat-related differentially expressed peridermal genes implied integrated activities of stress response and tissue development. Accumulating data suggest that the periderm, being a defensive tissue, responds to heat stress by enhancing the production and accumulation of periderm/skin layers to create a thick protective cover. Skin russeting may be a secondary outcome that develops upon continued expansion of the tuber: the inflexible skin cracks as new layers are produced below it.

It is reasonable to assume that the current potato cDNA array represents only a subset of genes expressed in the periderm; therefore, only limited heat-stress-related peridermal activities could be described here. A large number of clones that were hybridized to the periderm samples represented genes with no known function. Hence, the identification of transcription factor activities is of special interest, as these may be the key regulators, shedding light on the overall response.

The data presented here may also contribute to understanding the effect of seasonal variations and high temperature on periderm development in other plant systems, such as cork in woody species, root phellem and wound-healing. The rate of establishment of suberized periderm is an important factor in plant resistance to indirect damage following epidermal damage, such as water loss and pathogen invasion. However, the regulation, developmental stages and biosynthetic pathway of periderm development and suberization are still not completely understood. The direct isolation of peridermal genes and further characterization of phellogen activity would provide a better understanding and control of other agriculturally important issues, such as potato skin-set, melon netting, and tomato, apple, and pear russeting.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
 Supplementary data
References
 
The following supplementary data are available at JXB online.

Supplementary Fig. S1. Loop design diagram for microarray hybridizations.

Supplementary Table S1. List of S[H] differential genes.

Supplementary Table S2. List of Ph[H] differential genes.

Supplementary Table S3. List of SPh[H] differential genes.

Supplementary Table S4. Real-time PCR analysis of NAC/UBA-like reference gene.


   Acknowledgements
 
This research was supported by Research Grant No. IS-3581-04 from BARD, The United States–Israel Binational Agricultural Research and Development Fund, and is contribution No. 118/2009 from the ARO, The Volcani Center, Bet Dagan, Israel.


   Abbreviations
 
Ph[H], heat-treated phelloderm; S[H], heat-treated skin; SPh[H], heat-treated skin and phelloderm.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Concluding remarks
 Supplementary data
 References
 
Abeles F, Morgan P, Saltveit M. Ethylene in plant biology (1992) London, UK: Academic Press.

Ach RA, Taranto P, Gruissem W. A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and animals. The Plant Cell (1997) 9:1595–1606.[Abstract]

Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B-Methodological (1995) 57:289–300.

Barel G, Ginzberg I. Potato skin proteome is enriched with plant defence components. Journal of Experimental Botany (2008) 59:3347–3357.[Abstract/Free Full Text]

Boxall SF, Foster JM, Bohnert HJ, Cushman JC, Nimmo HG, Hartwell J. Conservation and divergence of circadian clock operation in a stress-inducible Crassulacean acid metabolism species reveals clock compensation against stress. Plant Physiology (2005) 137:969–982.[Abstract/Free Full Text]

Brenner ED, Lambert KN, Kaloshian I, Williamson VM. Characterization of LeMir, a root-knot nematode-induced gene in tomato with an encoded product secreted from the root. Plant Physiology (1998) 118:237–247.[Abstract/Free Full Text]

Chang S, Puryear J, Cairney J. A simple and efficient method for isolating RNA from pine trees. Plant Molecular Biology Reporter (1993) 11:113–116.[CrossRef]

Chen CH, Chen ZX. Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco. Plant Molecular Biology (2000) 42:387–396.[CrossRef][Web of Science][Medline]

Curaba J, Herzog M, Vachon G. GeBP, the first member of a new gene family in Arabidopsis, encodes a nuclear protein with DNA-binding activity and is regulated by KNAT1. The Plant Journal (2003) 33:305–317.[CrossRef][Medline]

de Jong H. Inheritance of russeting in cultivated diploid potatoes. Potato Research (1981) 24:309–313.[CrossRef][Web of Science]

Deng X, Phillips J, Meijer AH, Salamini F, Bartels D. Characterization of five novel dehydration-responsive homeodomain leucine zipper genes from the resurrection plant Craterostigma plantagineum. Plant Molecular Biology (2002) 49:601–610.[CrossRef][Web of Science][Medline]

Donnelly DJ, Coleman WK, Coleman SE. Potato microtuber production and performance: A review. American Journal of Potato Research (2003) 80:103–115.[CrossRef]

Doukhanina EV, Chen S, van der Zalm E, Godzik A, Reed J, Dickman MB. Identification and functional characterization of the BAG protein family in Arabidopsis thaliana. Journal of Biological Chemistry (2006) 281:18793–18801.[Abstract/Free Full Text]

Du LQ, Poovaiah BW. A novel family of Ca2+/calmodulin-binding proteins involved in transcriptional regulation: interaction with fsh/Ring3 class transcription activators. Plant Molecular Biology (2004) 54:549–569.[CrossRef][Web of Science][Medline]

Farage-Barhom S, Burd S, Sonego L, Perl-Treves R, Lers A. Expression analysis of the BFN1 nuclease gene promoter during senescence, abscission, and programmed cell death-related processes. Journal of Experimental Botany (2008) 59:3247–3258.[Abstract/Free Full Text]

Fujita T, Kouchi H, Ichikawa T, Syono K. Cloning of cDNAs for genes that are specifically or preferentially expressed during the development of tobacco genetic tumors. The Plant Journal (1994) 5:645–654.[CrossRef][Web of Science][Medline]

Gentleman RC, Carey VJ, Bates DM, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biology (2004) 5:R80.[CrossRef][Medline]

Gerchikov N, Keren-Keiserman A, Perl-Treves R, Ginzberg I. Wounding of melon fruits as a model system to study rind netting. Scientia Horticulturae (2008) 117:115–122.[CrossRef]

Harvey J, Lincoln J, Gilchrist D. Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an Agrobacterium rhizogenes-based functional screen. Molecular Genetics and Genomics (2008) 279:509–521.[CrossRef][Web of Science][Medline]

Helariutta Y, Fukaki H, Wysocka-Diller J, Nakajima K, Jung J, Sena G, Hauser MT, Benfey PN. The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signalling. Cell (2000) 101:555–567.[CrossRef][Web of Science][Medline]

Hwang J-U, Vernoud V, Szumlanski A, Nielsen E, Yang Z. A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex. Current Biology (2008) 18:1907–1916.[CrossRef][Web of Science][Medline]

Iwakawa H, Iwasaki M, Kojima S, Ueno Y, Soma T, Tanaka H, Semiarti E, Machida Y, Machida C. Expression of the ASYMMETRIC LEAVES2 gene in the adaxial domain of Arabidopsis leaves represses cell proliferation in this domain and is critical for the development of properly expanded leaves. The Plant Journal (2007) 51:173–184.[CrossRef][Medline]

Johansen DA. Plant microtechniques (1940) New York: McGraw-Hill Book Company, Inc.

Kisu Y, Ono T, Shimofurutani N, Suzuki M, Esaka M. Characterization and expression of a new class of zinc finger protein that binds to silencer region of ascorbate oxidase gene. Plant and Cell Physiology (1998) 39:1054–1064.[Abstract/Free Full Text]

Kolattukudy PE. Lipid polymers and associated phenols, their chemistry, biosynthesis and role in pathogenesis. Recent Advances in Phytochemistry (1977) 77:185–246.

Kolattukudy PE. Biochemistry and function of cutin and suberin. Canadian Journal of Botany (1984) 62:2918–2933.[CrossRef]

Kouchi H, Hata S. Isolation and characterization of novel nodulin cDNAs representing genes expressed at early stages of soybean nodule development. Molecular and General Genetics (1993) 238:106–119.

Krits P, Fogelman E, Ginzberg I. Potato steroidal glycoalkaloid levels and the expression of key isoprenoid metabolic genes. Planta (2007) 227:143–150.[CrossRef][Web of Science][Medline]

Kushwaha R, Singh A, Chattopadhyay S. Calmodulin7 plays an important role as transcriptional regulator in Arabidopsis seedling development. The Plant Cell (2008) 20:1747–1759.[Abstract/Free Full Text]

Lorkovic ZJ, Barta A. Genome analysis: RNA recognition motif (RRM) and K homology (KH) domain RNA-binding proteins from the flowering plant Arabidopsis thaliana. Nucleic Acids Research (2002) 30:623–635.[Abstract/Free Full Text]

Lulai EC, Freeman TP. The importance of phellogen cells and their structural characteristics in susceptibility and resistance to excoriation in immature and mature potato tuber (Solanum tuberosum L.) periderm. Annals of Botany (2001) 88:555–561.[Abstract/Free Full Text]

Niggeweg R, Thurow C, Kegler C, Gatz C. Tobacco transcription factor TGA2.2 is the main component of as-1-binding factor ASF-1 and is involved in salicylic acid- and auxin-inducible expression of as-1-containing target promoters. Journal of Biological Chemistry (2000) 275:19897–19905.[Abstract/Free Full Text]

Okazawa Y, Iriuda N. On occurrence of defected potato tubers with rough russeted skin. Japanese Journal of Crop Science (1980) 49:58–65.

Orzaez D, de Jong AJ, Woltering EJ. A tomato homologue of the human protein PIRIN is induced during programmed cell death. Plant Molecular Biology (2001) 46:459–468.[CrossRef][Web of Science][Medline]

Pysh LD, Wysocka-Diller JW, Camilleri C, Bouchez D, Benfey PN. The GRAS gene family in Arabidopsis: sequence characterization and basic expression analysis of the SCARECROW-LIKE genes. The Plant Journal (1999) 18:111–119.[CrossRef][Web of Science][Medline]

Reeve RM, Hautala E, Weaver ML. Anatomy and compositional variation within potatoes. 1. Developmental histology of the tuber. American Potato Journal (1969) 46:361–373.[CrossRef][Web of Science]

Rensink WA, Iobst S, Hart A, Stegalkina S, Liu J, Buell CR. Gene expression profiling of potato responses to cold, heat, and salt stress. Functional and Integrative Genomics (2005) 5:201–207.[CrossRef]

Ruzin SE. Plant microtechnique and microscopy (1999) New York: Oxford University Press.

Sabba RP, Lulai EC. Histological analysis of the maturation of native and wound periderm in potato (Solanum tuberosum L.) tuber. Annals of Botany (2002) 90:1–10.[Abstract/Free Full Text]

Sagasser M, Lu G-H, Hahlbrock K, Weisshaar B. Arabidopsis thaliana TRANSPARENT TESTA 1 is involved in seed coat development and defines the WIP subfamily of plant zinc finger proteins. Genes and Development (2002) 16:138–149.[Abstract/Free Full Text]

Smyth GK. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology (2004) 3. article 3.

Smyth GK, Michaud J, Scott HS. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics (2005) 21:2067–2075.[Abstract/Free Full Text]

Soderman E, Mattsson J, Engstrom P. The Arabidopsis homeobox gene ATHB-7 is induced by water deficit and by abscisic acid. The Plant Journal (1996) 10:375–381.[CrossRef][Web of Science][Medline]

Sugimoto K, Takeda S, Hirochika H. Transcriptional activation mediated by binding of a plant GATA-type zinc finger protein AGP1 to the AG-motif (AGATCCAA) of the wound-inducible Myb gene NtMyb2. The Plant Journal (2003) 36:550–564.[CrossRef][Web of Science][Medline]

Villavicencio LE, Blankenship SM, Yencho GC, Thomas JF, Raper CD. Temperature effect on skin adhesion, cell wall enzyme activity, lignin content, anthocyanins, growth parameters, and periderm histochemistry of sweetpotato. Journal of the American Society for Horticultural Science (2007) 132:729–738.[Abstract/Free Full Text]

Wu JQ, Hettenhausen C, Meldau S, Baldwin IT. Herbivory rapidly activates MAPK signalling in attacked and unattacked leaf regions but not between leaves of Nicotiana attenuata. The Plant Cell (2007) 19:1096–1122.[Abstract/Free Full Text]

Yamaguchi M, Timm H, Spurr AR. Effects of soil temperature on growth and nutrition of potato plants and tuberization, composition, and periderm structure of tubers. Proceedings of the American Society for Horticultural Science (1964) 84:412–423.

Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Research (2002) 30:e15.[Abstract/Free Full Text]

Yang YH, Speed T. Design issues for cDNA microarray experiments. Nature Reviews Genetics (2002) 3:579–588.[CrossRef][Web of Science][Medline]

Zanetti M, Terrile M, Arce D, Godoy A, San Segundo B, Casalongue C. Isolation and characterization of a potato cDNA corresponding to a 1-aminocyclopropane-1-carboxylate (ACC) oxidase gene differentially activated by stress. Journal of Experimental Botany (2002) 53:2455–2457.[Abstract/Free Full Text]


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