Plant Freezing Tolerance Research Articles

The effects of three levels of irrigation water on the improvement of cold tolerance of acclimated viola

Due to global climate changes, the growth of ornamental plants will be influenced by warming episodes in winter. The purpose of this examination was to estimate the effect of three levels of irrigation regimes on the freezing tolerance of acclimated viola (Viola × wittrockiana ‘Iona Gold with Blotch’). This experiments included three irrigation regimes (80, 60 and 40% FC) with different temperatures (20, 0, − 3, − 6, − 9, − 12, − 15, − 18, − 21 and − 24 °C). After irrigation regimes, proline and carbohydrate contents were recorded. Then, plants were moved to the thermo-gradient freezer and electrolyte leakage percentage (EL %), survival percentage (SU %), lethal temperature resulting in 50% mortality according to the electrolyte leakage percentage and survival percentage (LT50el and LT50su) were evaluated. The carbohydrate and proline content increased when plants were under drought stress conditions. Electrolyte leakage percentage across three levels of irrigation treatment increased by lowering the temperature; this increase at − 24 °C was fivefold more than the control. Survival percentage in all treatments was not affected in the thermal ranges of 0 to − 9 °C, but decreasing the temperature to − 24 °C resulted in the full loss in all treatments. Vegetative growth and reproductive components were significantly affected by drought and freezing temperature treatments. Dry weights of different parts of plants reached peak values when plants were under 60% FC at 0 °C condition. A negative correlation between LT50el with carbohydrate and proline levels was found and drought treatment significantly increased freezing tolerance (lower LT50) in plants grown under 60% FC compared to other treatments.

The cold response regulator CBF1 promotes Arabidopsis hypocotyl growth at ambient temperatures.

Light and temperature are two core environmental factors that coordinately regulate plant growth and survival throughout their entire life cycle. However, the mechanisms integrating light and temperature signaling pathways in plants remain poorly understood. Here, we report that CBF1, an AP2/ERF-family transcription factor essential for plant cold acclimation, promotes hypocotyl growth under ambient temperatures in Arabidopsis. We show that CBF1 increases the protein abundance of PIF4 and PIF5, two phytochrome-interacting bHLH-family transcription factors that play pivotal roles in modulating plant growth and development, by directly binding to their promoters to induce their gene expression, and by inhibiting their interaction with phyB in the light. Moreover, our data demonstrate that CBF1 promotes PIF4/PIF5 protein accumulation and hypocotyl growth at both 22°C and 17°C, but not at 4°C, with a more prominent role at 17°C than at 22°C. Together, our study reveals that CBF1 integrates light and temperature control of hypocotyl growth by promoting PIF4 and PIF5 protein abundance in the light, thus providing insights into the integration mechanisms of light and temperature signaling pathways in plants.

Photosynthesis Characteristics of Tall Fescue under Snow-Melting Agent, Acid Precipitation and Freeze-Thaw Stress

To explore the physiological response characteristics of plant photosynthesis under acid precipitation stress (A stress), snow-melting agent stress (S stress), and freeze-thaw stress (F stress) as well as their various combinations, the net assimilation rate (NAR), intercellular CO2 concentration (Ci), stomatal conductance (Gs) and water use efficiency (WUE) of tall fescue (Festuca arundinacea Schreb.) under these three stresses respectively and four kinds of combined stresses were measured. The effects of these different kinds of stresses on the photosynthesis parameters were then evaluated, and the major mechanisms of main factors affecting the interactions between stresses were speculated. The experimental results indicated that both NAR and WUE decreased significantly in response to all stresses and Ci increased dramatically in response to all stresses, while Gs ascended under F stress but descended in response to A stress and S stress. The photosynthetic efficiency significantly decreased as the temperature decreased and did not clearly recover as the temperature increased. Moreover, the combination of A stress and S stress had fewer effects on photosynthesis than did the S stress alone, while the effects resulting from F stress were similar at low temperature. The major controlling mechanism is most likely the strong inhibition of photosystem II and the promotion of antioxidant enzymes resulting from acid precipitation as well as NaCl may strengthen the freezing tolerance of plants.

Cold-Induced CBF–PIF3 Interaction Enhances Freezing Tolerance by Stabilizing the phyB Thermosensor in Arabidopsis

Growth inhibition and cold-acclimation strategies help plants withstand cold stress, which adversely affects growth and survival. PHYTOCHROME B (phyB) regulates plant growth through perceiving both light and ambient temperature signals. However, the mechanism by which phyB mediates the plant response to cold stress remains elusive. Here, we show that the key transcription factors mediating cold acclimation, C-REPEAT BINDING FACTORs (CBFs), interact with PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) under cold stress, thus attenuating the mutually assured destruction of PIF3–phyB. Cold-stabilized phyB acts downstream of CBFs to positively regulate freezing tolerance by modulating the expression of stress-responsive and growth-related genes. Consistent with this, phyB mutants exhibited a freezing-sensitive phenotype, whereas phyB-overexpression transgenic plants displayed enhanced freezing tolerance. Further analysis showed that the PIF1, PIF4, and PIF5 proteins, all of which negatively regulate plant freezing tolerance, were destabilized by cold stress in a phytochrome-dependent manner. Collectively, our study reveals that CBFs–PIF3–phyB serves as an important regulatory module for modulating plant response to cold stress.

Combined transcriptomic and metabolomic analyses uncover rearranged gene expression and metabolite metabolism in tobacco during cold acclimation

Cold temperatures often severely restrict the growth, distribution and productivity of plants. The freezing tolerance of plants from temperate climates can be improved by undergoing periods of cold acclimation (CA). Tobacco is an important economic plant and is sensitive to cold stress. However, the dynamic changes and regulatory mechanisms of gene expression and metabolic processes during CA remain largely unknown. In this study, we performed RNA sequencing and metabolomic profiling analyses to identify the genes and metabolites specifically expressed during CA. Our transcriptomic data revealed 6905 differentially expressed genes (DEGs) during CA. Functional annotation and enrichment analyses revealed that the DEGs were involved mainly in signal transduction, carbohydrate metabolism and phenylpropanoid biosynthesis. Moreover, a total of 35 significantly changed metabolites were identified during CA via an LC-MS platform. Many protective metabolites, such as amino acids, carbohydrates, tricarboxylic acid (TCA) cycle intermediates and phenylpropanoid-related substances, were identified during CA. The gene-metabolite network extensively outlined the biological processes associated with the utilization of sugars, activation of amino acid metabolism, TCA cycle and phenylpropanoid biosynthesis in tobacco under CA. The results of our present study provide a comprehensive view of signal transduction and regulation, gene expression and dynamic changes in metabolites during CA.

Identification of tissue-specific and cold-responsive lncRNAs in Medicago truncatula by high-throughput RNA sequencing

BackgroundLong non-coding RNAs (lncRNAs) play important roles in the regulation of plant responses to environmental stress by acting as essential regulators of gene expression. However, whether and how lncRNAs are involved in cold acclimation-dependent freezing tolerance in plants remains largely unknown. Medicago truncatula is a prominent model for studies of legume genomics, and distinguished by its cold-acclimation characteristics. To determine the roles of lncRNAs in plant cold stress response, we conducted genome-wide high-throughput sequencing in the legume model plant M. truncatula.ResultsRNA-seq data were generated from twelve samples for the four treatments, i.e., non-cold treated leaves and roots, cold-treated leaves and roots of M. truncatula Jemalong A17 seedlings. A total of 1204 million raw reads were generated. Of them, 1150 million filtered reads after quality control (QC) were subjected to downstream analysis. A large number of 24,368 unique lncRNAs were identified from the twelve samples. Among these lncRNAs, 983 and 1288 were responsive to cold treatment in the leaves and roots, respectively. We further found that the intronic-lncRNAs were most sensitive to the cold treatment. The cold-responsive lncRNAs were unevenly distributed across the eight chromosomes in M. truncatula seedlings with obvious preferences for locations. Further analyses revealed that the cold-responsive lncRNAs differed between leaves and roots. The putative target genes of the lncRNAs were predicted to mainly involve the processes of protein translation, transport, metabolism and nucleic acid transcription. Furthermore, the networks of a tandem array of CBF/DREB1 genes that were reported to be located in a major freezing tolerance QTL region on chromosome 6 and their related lncRNAs were dissected based on their gene expression and chromosome location.ConclusionsWe identified a comprehensive set of lncRNAs that were responsive to cold treatment in M. truncatula seedlings, and discovered tissue-specific cold-responsive lncRNAs in leaves and roots. We further dissected potential regulatory networks of CBF Intergenic RNA (MtCIR1) and MtCBFs that play critical roles in response and adaptation of M. truncatula to cold stress.

A metabolomics study of ascorbic acid-induced in situ freezing tolerance in spinach (Spinacia oleracea L.).

Freeze–thaw stress is one of the major environmental constraints that limit plant growth and reduce productivity and quality. Plants exhibit a variety of cellular dysfunctions following freeze–thaw stress, including accumulation of reactive oxygen species (ROS). This means that enhancement of antioxidant capacity by exogenous application of antioxidants could potentially be one of the strategies for improving freezing tolerance (FT) of plants. Exogenous application of ascorbic acid (AsA), as an antioxidant, has been shown to improve plant tolerance against abiotic stresses but its effect on FT has not been investigated. We evaluated the effect of AsA‐feeding on FT of spinach (Spinacia oleracea L.) at whole plant and excised‐leaf level, and conducted metabolite profiling of leaves before and after AsA treatment to explore metabolic explanation for change in FT. AsA application did not impede leaf growth, instead slightly promoted it. Temperature‐controlled freeze–thaw tests revealed AsA‐fed plants were more freezing tolerant as indicated by: (a) less visual damage/mortality; (b) lower ion leakage; and (c) less oxidative injury, lower abundance of free radicals (O2·- and H2O2). Comparative leaf metabolite profiling revealed clear separation of metabolic phenotypes for control versus AsA‐fed leaves. Specifically, AsA‐fed leaves had greater abundance of antioxidants (AsA, glutathione, alpha‐ & gamma‐tocopherol) and compatible solutes (proline, galactinol, and myo‐inositol). AsA‐fed leaves also had higher activity of antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, and catalase). These changes, together, may improve FT via alleviating freeze‐induced oxidative stress as well as protecting membranes from freeze desiccation. Additionally, improved FT by AsA‐feeding may potentially include enhanced cell wall/lignin augmentation and bolstered secondary metabolism as indicated by diminished level of phenylalanine and increased abundance of branched amino acids, respectively.

The SWI/SNF ATP-Dependent Chromatin Remodeling Complex in Arabidopsis Responds to Environmental Changes in Temperature-Dependent Manner.

SWI/SNF ATP-dependent chromatin remodeling complexes (CRCs) play important roles in the regulation of transcription, cell cycle, DNA replication, repair, and hormone signaling in eukaryotes. The core of SWI/SNF CRCs composed of a SWI2/SNF2 type ATPase, a SNF5 and two of SWI3 subunits is sufficient for execution of nucleosome remodeling in vitro. The Arabidopsis genome encodes four SWI2/SNF2 ATPases, four SWI3, a single SNF5 and two SWP73 subunits. Genes of the core SWI/SNF components have critical but not fully overlapping roles during plant growth, embryogenesis, and sporophyte development. Here we show that the Arabidopsis swi3c mutant exhibits a phenotypic reversion when grown at lower temperature resulting in partial restoration of its embryo, root development and fertility defects. Our data indicates that the swi3c mutation alters the expression of several genes engaged in low temperature responses. The location of SWI3C-containing SWI/SNF CRCs on the ICE1, MYB15 and CBF1 target genes depends on the temperature conditions, and the swi3c mutation thus also influences the transcription of several cold-responsive (COR) genes. These findings, together with genetic analysis of swi3c/ice1 double mutant and enhanced freezing tolerance of swi3c plants illustrate that SWI/SNF CRCs contribute to fine-tuning of plant growth responses to different temperature regimes.

Analysis of Changes in Plant Cell Wall Composition and Structure During Cold Acclimation.

The cell wall has a crucial influence on the mechanical properties of plant cells. It therefore has a strong impact on the freezing behavior and very likely also the freezing tolerance of plants. However, not many studies have addressed the question how cell wall composition and structure impact plant freezing tolerance and cold acclimation. In this chapter, we describe a comprehensive workflow to extract total cell wall material from leaves of Arabidopsis thaliana and to separate this material into fractions enriched in crystalline cellulose, pectins, and hemicelluloses by sequential fractionation. We further describe methods for the analysis of chemical structure, monosaccharide composition, and cellulose and uronic acid contents in the total cell wall material and the fractions in response to cold acclimation. Structural properties of cell wall material are analyzed by attenuated total reflectance-Fourier-transform infrared spectrometry (ATR-FTIR) and monosaccharide composition by gas chromatography-mass spectrometry (GC-MS) after isolation of alditol acetate derivatives of the sugars.

Subcellular Compartmentation of Metabolites Involved in Cold Acclimation.

Plant cells are heavily compartmentalized, and metabolite concentrations in the various compartments differ significantly. Thus, determination of metabolite abundance in whole-cell extracts may be misleading, when the role of a compound in plant freezing tolerance shall be evaluated. Here, we describe a method for the separation of the largest compartments of plant cells, the vacuole, plastid, and cytosol. With more elaborate analysis, this method can be expanded to also resolve mitochondria and other compartments.

Measuring Freezing Tolerance of Leaves and Rosettes: Electrolyte Leakage and Chlorophyll Fluorescence Assays.

Quantitative assessment of freezing tolerance is essential to unravel plant adaptations to cold temperatures. Not only the survival of whole plants, but also impairment of detached leaves or small rosettes after a freeze-thaw cycle can be used to accurately quantify plant freezing tolerance in terms of LT50 values. Here we describe two methods to determine the freezing tolerance of detached leaves or rosettes using a full or selected set of freezing temperatures and an additional method using chlorophyll fluorescence as a different physiological parameter. Firstly, we illustrate how to assess the integrity of (predominantly) the plasma membrane during freezing using an electrolyte leakage assay. Secondly, we provide a chlorophyll fluorescence imaging protocol to determine the freezing tolerance of the photosynthetic apparatus.

Introduction: Plant Cold Acclimation and Winter Survival.

This introductory chapter provides a brief overview of plant freezing tolerance, cold acclimation, including subzero acclimation, and the subsequent deacclimation when plants return to warm conditions favoring growth and development. We describe the basic concepts and approaches that are currently followed to investigate these phenomena. We highlight the multidisciplinary nature of these investigations and the necessity to use methodologies from different branches of science, such as ecology, genetics, physiology, cell biology, biochemistry, and biophysics to gain a complete understanding of the complex adaptive mechanisms ultimately underlying plant winter survival.

MUR1-mediated cell-wall fucosylation is required for freezing tolerance in Arabidopsis thaliana.

Summary Forward genetic screens play a key role in the identification of genes contributing to plant stress tolerance. Using a screen for freezing sensitivity, we have identified a novel freezing tolerance gene, SENSITIVE‐TO‐FREEZING8, in Arabidopsis thaliana. We identified SFR8 using recombination‐based mapping and whole‐genome sequencing. As SFR8 was predicted to have an effect on cell wall composition, we used GC‐MS and polyacrylamide gel electrophoresis to measure cell‐wall fucose and boron (B)‐dependent dimerization of the cell‐wall pectic domain rhamnogalacturonan II (RGII) in planta. After treatments to promote borate‐bridging of RGII, we assessed freeze‐induced damage in wild‐type and sfr8 plants by measuring electrolyte leakage from freeze‐thawed leaf discs.We mapped the sfr8 mutation to MUR1, a gene encoding the fucose biosynthetic enzyme GDP‐d‐mannose‐4,6‐dehydratase. sfr8 cell walls exhibited low cell‐wall fucose levels and reduced RGII bridging. Freezing sensitivity of sfr8 mutants was ameliorated by B supplementation, which can restore RGII dimerization. B transport mutants with reduced RGII dimerization were also freezing‐sensitive.Our research identifies a role for the structure and composition of the plant primary cell wall in determining basal plant freezing tolerance and highlights the specific importance of fucosylation, most likely through its effect on the ability of RGII pectin to dimerize.

Season specificity in the cold-induced calcium signal and the volatile chemicals in the atmosphere.

Cold-induced Ca2+ signals in plants are widely accepted to be involved in cold acclimation. Surprisingly, despite using Arabidopsis plants grown in a growth chamber, we observed a clear seasonal change in cold-induced Ca2+ signals only in roots. Ca2+ signals were captured using Arabidopsis expressing Yellow Cameleon 3.60. In winter, two Ca2+ signal peaks were observed during a cooling treatment from 20 to 0°C, but in summer only one small peak was observed under the same cooling condition. In the spring and autumn seasons, an intermediate type of Ca2+ signal, which had a delayed first peak and smaller second peaks compared with the those of the winter type, was observed. Volatile chemicals and/or particles in the air from the outside may affect plants in the growth chamber. This idea is supported by the fact that incubation of plants with activated carbon changed the intermediate-type Ca2+ signal to the summer-type. The seasonality was also observed in the freezing tolerance of plants cold-acclimated in a low-temperature chamber. The solar radiation intensity was weakly correlated, not only with the seasonal characteristics of the Ca2+ signal but also with freezing tolerance. It has been reported that the ethylene concentration in the atmosphere seasonally changes depending on the solar radiation intensity. Ethylene gas and 1-aminocyclopropane-1-carboxylic acid treatment affected the Ca2+ signals, the shape of which became a shape close to, but not the same as, the winter type from the other types, indicating that ethylene may be one of several factors influencing the cold-induced Ca2+ signal.

PUB25 and PUB26 Promote Plant Freezing Tolerance by Degrading the Cold Signaling Negative Regulator MYB15.

Cold stress adversely affects plant growth, development, and crop productivity and quality. Plants employ cold acclimation strategy to protect them from cold damage. The transcription-factor-CBF-dependent cold signaling pathway plays a key role in plant cold acclimation. However, how this signaling pathway is dynamically and precisely regulated remains unknown. Here, we report that two U-box type E3 ubiquitin ligases, PUB25 and PUB26, positively regulate freezing tolerance in Arabidopsis thaliana. Both PUB25 and PUB26 poly-ubiquitinate MYB15, a transcriptional repressor of the CBF-dependent cold signaling pathway, leading to MYB15 degradation and thus enhanced CBF expression under cold stress. Furthermore, cold-activated OST1 specifically phosphorylates PUB25 and PUB26 at conserved threonine residues, enhancing their E3 activity and facilitating the cold-induced degradation of MYB15. Our results thus unravel the regulatory role of the OST1-PUB25/26 module in regulating the duration and amplitude of the cold response by controlling the homeostasis of the negative regulator MYB15.

Type II Ice-Binding Proteins Isolated from an Arctic Microalga Are Similar to Adhesin-Like Proteins and Increase Freezing Tolerance in Transgenic Plants.

Microalgal ice-binding proteins (IBPs) in the polar region are poorly understood at the genome-wide level, although they are important for cold adaptation. Through the transcriptome study with the Arctic green alga Chloromonas sp. KNF0032, we identified six Chloromonas IBP genes (CmIBPs), homologous with the previously reported IBPs from Antarctic snow alga CCMP681 and Antarctic Chloromonas sp. They were organized with multiple exon/intron structures and low-temperature-responsive cis-elements in their promoters and abundantly expressed at low temperature. The biological functions of three representative CmIBPs (CmIBP1, CmIBP2 and CmIBP3) were tested using in vitro analysis and transgenic plant system. CmIBP1 had the most effective ice recrystallization inhibition (IRI) activities in both in vitro and transgenic plants, and CmIBP2 and CmIBP3 had followed. All transgenic plants grown under nonacclimated condition were freezing tolerant, and especially 35S::CmIBP1 plants were most effective. After cold acclimation, only 35S::CmIBP2 plants showed slightly increased freezing tolerance. Structurally, the CmIBPs were predicted to have β-solenoid forms with parallel β-sheets and repeated TXT motifs. The repeated TXT structure of CmIBPs appears similar to the AidA domain-containing adhesin-like proteins from methanogens. We have shown that the AidA domain has IRI activity as CmIBPs and phylogenetic analysis also supported that the AidA domains are monophyletic with ice-binding domain of CmIBPs, and these results suggest that CmIBPs are a type of modified adhesins.

A Role for PICKLE in the Regulation of Cold and Salt Stress Tolerance in Arabidopsis.

Arabidopsis PICKLE (PKL) is a putative CHD3-type chromatin remodeling factor with important roles in regulating plant growth and development as well as RNA-directed DNA methylation (RdDM). The role of PKL protein in plant abiotic stress response is still poorly understood. Here, we report that PKL is important for cold stress response in Arabidopsis. Loss-of-function mutations in the PKL gene lead to a chlorotic phenotype in seedlings under cold stress, which is caused by the alterations in the transcript levels of some chlorophyll metabolism-related genes. The pkl mutant also exhibits increased electrolyte leakage after freezing treatment. These results suggest that PKL is required for proper chilling and freezing tolerance in plants. Gene expression analysis shows that CBF3, encoding a key transcription factor involved in the regulation of cold-responsive genes, exhibits an altered transcript level in the pkl mutant under cold stress. Transcriptome data also show that PKL regulates the expression of a number of cold-responsive genes, including RD29A, COR15A, and COR15B, possibly through its effect on the expression of CBF3 gene. Mutation in PKL gene also results in decreased cotyledon greening rate and reduced primary root elongation under high salinity. Together, our results suggest that PKL regulates plant responses to cold and salt stress.

BYPASS1-LIKE, A DUF793 Family Protein, Participates in Freezing Tolerance via the CBF Pathway in Arabidopsis.

The C-REPEAT BINDING FACTOR signaling pathway is strictly modulated by numerous factors and is essential in the cold response of plants. Here, we show that the DUF793 family gene BYPASS1-LIKE modulates freezing tolerance through the CBFs in Arabidopsis. The expression of B1L was rapidly induced under cold treatment. Comparing to wild type, B1L knockout mutants were more sensitive to freezing treatment, whereas B1L-overexpressing lines were more tolerant. The expression of CBFs and CBF target genes was significantly decreased in b1l mutant. Using yeast two-hybrid screening system, 14-3-3λ was identified as one of proteins interacting with B1L. The interaction was confirmed with bimolecular fluorescence complementation assay and co-immunoprecipitation assay. Biochemical assays revealed that b1l mutation promoted the degradation of CBF3 compared to wild type, whereas 14-3-3κλ mutant and b1l 14-3-3κλ mutant suppressed the degradation of CBF3. Consistently, 14-3-3κλ and b1l 14-3-3κλ mutants showed enhanced freezing tolerance compared to wild type. These results indicate that B1L enhances the freezing tolerance of plants, at least partly through stabilizing CBF. Our findings improve our understanding of the regulation of CBF in response to cold stress.

Identification and comparative analysis of the CIPK gene family and characterization of the cold stress response in the woody plant Prunus mume.

Prunus mume is an important ornamental woody plant that grows in tropical and subtropical regions. Freezing stress can adversely impact plant productivity and limit the expansion of geographical locations. Understanding cold-responsive genes could potentially bring about the development of new ways to enhance plant freezing tolerance. Members of the serine/threonine protein kinase (CIPK) gene family play important roles in abiotic stress. However, the function of CIPK genes in P. mume remains poorly defined. A total of 16 CIPK genes were first identified in P. mume. A systematic phylogenetic analysis was conducted in which 253 CIPK genes from 12 species were divided into three groups. Furthermore, we analysed the chromosomal locations, molecular structures, motifs and domains of CIPK genes in P. mume. All of the CIPK sequences had NAF domains and promoter regions containing cis-acting regulatory elements of the related stress response. Three PmCIPK genes were identified as Pmu-miR172/167-targeted sites. Transcriptome data showed that most PmCIPK genes presented tissue-specific and time-specific expression profiles. Nine genes were highly expressed in flower buds in December and January, and 12 genes were up-regulated in stems in winter. The expression levels of 12 PmCIPK genes were up-regulated during cold stress treatment confirmed by qRT-PCR. Our study improves understanding of the role of the PmCIPK gene family in the low temperature response in woody plants and provides key candidate genes and a theoretical basis for cold resistance molecular-assisted breeding technology in P. mume.

Both cold and sub-zero acclimation induce cell wall modification and changes in the extracellular proteome in Arabidopsis thaliana

Cold acclimation (CA) leads to increased plant freezing tolerance during exposure to low, non-freezing temperatures as a result of many physiological, biochemical and molecular changes that have been extensively investigated. In addition, many plant species, such as Arabidopsis thaliana, respond to a subsequent exposure to mild, non-damaging freezing temperatures with an additional increase in freezing tolerance referred to as sub-zero acclimation (SZA). There is comparatively little information available about the molecular basis of SZA. However, previous transcriptomic studies indicated that cell wall modification may play an important role during SZA. Here we show that CA and SZA are accompanied by extensive changes in cell wall amount, composition and structure. While CA leads to a significant increase in cell wall amount, the relative proportions of pectin, hemicellulose and cellulose remained unaltered during both CA and SZA. However, both treatments resulted in more subtle changes in structure as determined by infrared spectroscopy and monosaccharide composition as determined by gas chromatography-mass spectrometry. These differences could be related through a proteomic approach to the accumulation of cell wall modifying enzymes such as pectin methylesterases, pectin methylesterase inhibitors and xyloglucan endotransglucosylases/hydrolases in the extracellular matrix.