Changes of Detergent-Resistant Plasma Membrane Proteins in Oat and Rye during Cold Acclimation: Association with Differential Freezing Tolerance

Cold acclimation (CA) results in an increase in freezing tolerance of plants, which is closely associated to functional changes of the plasma membrane (PM). Although proteomic studies have revealed compositional changes of the PM during CA, there has been no large-scale study of how the microdomains in the PM, which contains specific lipids and proteins, change during CA. Therefore, we conducted semiquantitative shotgun proteomics using microdomain-enriched detergent-resistant membrane (DRM) fractions extracted from low freezing-tolerant oat and highly freezing-tolerant rye. We identified 740 and 809 DRM proteins in oat and rye, respectively. Among the proteins identified, the abundances of a variety of proteins, such as P-type ATPase and aquaporins, were affected by CA in both oat and rye. Some CA-responsive proteins in the DRM fractions, such as heat shock protein 70, changed differently in oat and rye. In addition, changes in lipocalins and sugar transporters in the DRM fractions were different from those found in total PM fraction during CA. This is the first report to describe compositional changes in the DRM during CA. The proteomic profiles obtained in the present study hint at many possible microdomain functions associated with CA and freezing tolerance

Changes of Detergent-Resistant Plasma Membrane Proteins in Oat and Rye during Cold Acclimation: Association with Differential Freezing Tolerance

Cold acclimation (CA) results in an increase in freezing tolerance of plants, which is closely associated to functional changes of the plasma membrane (PM). Although proteomic studies have revealed compositional changes of the PM during CA, there has been no large-scale study of how the microdomains in the PM, which contains specific lipids and proteins, change during CA. Therefore, we conducted semiquantitative shotgun proteomics using microdomain-enriched detergent-resistant membrane (DRM) fractions extracted from low freezing-tolerant oat and highly freezing-tolerant rye. We identified 740 and 809 DRM proteins in oat and rye, respectively. Among the proteins identified, the abundances of a variety of proteins, such as P-type ATPase and aquaporins, were affected by CA in both oat and rye. Some CA-responsive proteins in the DRM fractions, such as heat shock protein 70, changed differently in oat and rye. In addition, changes in lipocalins and sugar transporters in the DRM fractions were different from those found in total PM fraction during CA. This is the first report to describe compositional changes in the DRM during CA. The proteomic profiles obtained in the present study hint at many possible microdomain functions associated with CA and freezing tolerance

Changes of Detergent-Resistant Plasma Membrane Proteins in Oat and Rye during Cold Acclimation: Association with Differential Freezing Tolerance

Cold acclimation (CA) results in an increase in freezing tolerance of plants, which is closely associated to functional changes of the plasma membrane (PM). Although proteomic studies have revealed compositional changes of the PM during CA, there has been no large-scale study of how the microdomains in the PM, which contains specific lipids and proteins, change during CA. Therefore, we conducted semiquantitative shotgun proteomics using microdomain-enriched detergent-resistant membrane (DRM) fractions extracted from low freezing-tolerant oat and highly freezing-tolerant rye. We identified 740 and 809 DRM proteins in oat and rye, respectively. Among the proteins identified, the abundances of a variety of proteins, such as P-type ATPase and aquaporins, were affected by CA in both oat and rye. Some CA-responsive proteins in the DRM fractions, such as heat shock protein 70, changed differently in oat and rye. In addition, changes in lipocalins and sugar transporters in the DRM fractions were different from those found in total PM fraction during CA. This is the first report to describe compositional changes in the DRM during CA. The proteomic profiles obtained in the present study hint at many possible microdomain functions associated with CA and freezing tolerance

DataSheet_1_Cold acclimation is affected by diurnal cycles and minute-scale random temperature fluctuations via calcium signals.pdf

Molecular and physiological processes during cold acclimation (CA) have been investigated using plants incubated under constant low-temperature conditions. However, to comprehensively characterize CA in the field, the effects of day–night temperature cycles and minute-scale random temperature fluctuations must be clarified. Thus, we developed an experimental system that can maintain diurnal cycles and random temperature fluctuations during CA treatments. On the basis of the temperature changes in the field, three CA conditions were applied: conventional CA at 2°C (con-CA), CA with a 10°C day/2°C night cycle (C-CA), and C-CA with random temperature fluctuations only during the day (FC-CA). Because cold-induced Ca2+ signals help regulate CA, the effects of Ca2+ signals during the three CA treatments were examined using Ca2+ channel blockers (LaCl3 and ruthenium red). The freezing tolerance of Arabidopsis thaliana was similar after the C-CA and con-CA treatments, but it decreased following the FC-CA treatment. The analysis of transcription factors regulating CA processes indicated CBF/DREB1 expression levels tended to be highest for the con-CA treatment, followed by the FC-CA and C-CA treatments. Moreover, the Ca2+ signals substantially contributed to the freezing tolerance of the plants that underwent the FC-CA and C-CA treatments, while also considerably modulating gene expression in the FC-CA-treated plants. Furthermore, the Ca2+ signals enhanced CBF/DREB1 expression during the FC-CA treatment, but the Ca2+ signals derived from intracellular organelles suppressed the expression of CBF2/DREB1C and CBF3/DREB1A during the C-CA treatment. Thus, diurnal temperature cycles and random temperature fluctuations affect CA through different calcium signals, implying that plants regulate CA by precisely sensing temperature changes in the field.</p

Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC–MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (<i>Arabidopsis</i> and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells

MALDI-IMS/MS analysis of ions at <i>m/z</i> 419 and <i>m/z</i> 449 on longitudinal or transverse sections of a black rice seed.

<p>A–E, Longitudinal section images. Scale bar: 1.0 mm. F–J, Transverse section images. Upper side in transverse sections is the dorsal side of the black rice seed. Scale bar: 0.5 mm. A, F, Optical images. B, G, Localization pattern of the fragment ions at <i>m/z</i> 287, derived from the precursor ions at <i>m/z</i> 449, corresponding to cyanidin-3-<i>O</i>-hexoside. C, H, Localization pattern of the fragment ions at <i>m/z</i> 287, derived from the precursor ions at <i>m/z</i> 419, corresponding to cyanidin-3-<i>O</i>-pentoside. D, I, Localization pattern of the fragment ions at <i>m/z</i> 317, derived from the precursor ions at <i>m/z</i> 449, corresponding to petunidin-3-<i>O</i>-pentoside. E, J, Merged ion image of <i>m/z</i> 449→287 (red), <i>m/z</i> 419→287 (green), and <i>m/z</i> 449→317 (blue). K, Four regions defined in the intensity quantification of each fragment ion. L–N, Relative intensity of the fragment ions corresponding to cyanidin-3-<i>O</i>-hexoside (L), cyanidin-3-<i>O</i>-pentoside (M), and petunidin-3-<i>O</i>-pentoside (N). AV: anterior ventral region, PV: posterior ventral region, PD: posterior dorsal region, AD: anterior dorsal region. Data was collected from 7 sections of 3 black rice seeds (means ± S.E.). Different denote significant differences among groups means from a Tukey-Kramer test (<i>p</i><0.01). O, HPLC chromatogram of anthocyanins in the black rice crude extract. Peak 1 and 2, unknown; peak 3, cyanidin-3-<i>O</i>-glucoside; peak 4, peonidin-3-<i>O</i>-glucoside. P, Q, Contents of cyanidin-3-<i>O</i>-glucoside (P) and peonidin-3-<i>O</i>-glucoside (Q) in each part of black rice seeds determined by HPLC and expressed in nmol per mg of seed pieces ± S.E in triplicate.</p

MALDI-IMS analysis of anthocyanins in black rice sections.

<p>A, Black rice section stained with toluidine blue O after MALDI-IMS analysis of the ions at <i>m/z</i> 449. B, Localization pattern of the ions at <i>m/z</i> 449. C, Localization pattern of the ions at <i>m/z</i> 822 corresponding to PC (diacyl C36:3), which marks the nucellus epidermis/aleurone layer. D, Localization pattern of the ions at <i>m/z</i> 496 corresponding to LPC (C16:0), which marks the endosperm. E, Merged ion image of <i>m/z</i> 449 (red), <i>m/z</i> 822 (green) and <i>m/z</i> 496 (white). F, Black rice section stained with toluidine blue O after MALDI-IMS analysis of the ions at <i>m/z</i> 463. G, Localization pattern of the ions at <i>m/z</i> 463. H, Localization pattern of the ions at <i>m/z</i> 822 corresponding to PC (diacyl C36:3). I, Localization pattern of the ions at <i>m/z</i> 496 corresponding to LPC (C16:0). J, Merged ion image of <i>m/z</i> 463 (red), <i>m/z</i> 822 (green) and <i>m/z</i> 496 (white). Scale bar: 1.0 mm.</p

Identified anthocyanin species in this study.

<p>*Identified only by MALDI-IMS/MS on black rice tissue.</p

MALDI-IMS/MS analysis of the ions at <i>m/z</i> 449 and <i>m/z</i> 463.

<p>A, B, MS/MS spectrum of ions at <i>m/z</i> 449 (A) and <i>m/z</i> 463 (B) obtained from the pericarp of a black rice section. C, Optical image of a black rice section after analysis of the ions at <i>m/z</i> 449. D, E, Localization patterns of the fragment ions at <i>m/z</i> 287 (E) and 317 (F), derived from the parent ions at <i>m/z</i> 449, corresponding to cyanidin-3-<i>O</i>-hexoside and petunidin-3-<i>O</i>-pentoside, respectively. F, Localization pattern of the ions at <i>m/z</i> 822 corresponding to PC (diacyl C36:3), which marks the nucellus epidermis/aleurone layer. G, Merged ion image of <i>m/z</i> 449→287 (red), <i>m/z</i> 449→317 (green) and <i>m/z</i> 822 (blue). H, Optical image of a black rice section after analysis of the ions at <i>m/z</i> 463. I, J, Localization patterns of the fragment ions at <i>m/z</i> 301 (I) and <i>m/z</i> 331 (J), derived from parent ions at <i>m/z</i> 463, corresponding to peonidin-3-<i>O</i>-hexoside and malvidin-3-<i>O</i>-pentoside, respectively. K, Localization pattern of the ions at <i>m/z</i> 822 corresponding to PC (diacyl C36:3). L, Merged ion image of <i>m/z</i> 463→301 (red), <i>m/z</i> 463→331 (green), and <i>m/z</i> 822 (blue). Scale bar: 1.0 mm.</p

Detergent-resistant Plasma Membrane Proteome in Oat and Rye: Similarities and Dissimilarities between Two Monocotyledonous Plants

The plasma membrane (PM) is involved in important cellular processes that determine the growth, development, differentiation, and environmental signal responses of plant cells. Some of these dynamic reactions occur in specific domains in the PM. In this study, we performed comparable nano-LC–MS/MS-based large-scale proteomic analysis of detergent-resistant membrane (DRM) fractions prepared from the PM of oat and rye. A number of proteins showed differential accumulation between the PM and DRM, and some proteins were only found in the DRM. Numerous proteins were identified as DRM proteins in oat (219 proteins) and rye (213 proteins), of which about half were identified only in the DRM. The DRM proteins were largely common to those found in dicotyledonous plants (<i>Arabidopsis</i> and tobacco), which suggests common functions associated with the DRM in plants. Combination of semiquantitative proteomic analysis and prediction of post-translational protein modification sites revealed differences in several proteins associated with the DRM in oat and rye. It is concluded that protein distribution in the DRM is unique from that in the PM, partly because of the physicochemical properties of the proteins, and the unique distribution of these proteins may define the functions of the specific domains in the PM in various physiological processes in plant cells