Cytotoxic abietane diterpenoids from Salvia leriifolia Benth.

The Phytochemical profiling of the root extract of Salvia leriifolia, an endemic plant of Iran, was investigated and 16 abietane diterpenes were isolated, and three were original compounds. 1D and 2D NMR and HRMS performed structural elucidation. The absolute configuration of the previously unreported compounds was determined by circular dichroism (ECD). The cytotoxicity of the isolated compounds was investigated against AGS, MIA PaCa-2, HeLa, and MCF-7 cell lines by the MTT assay. The known diterpene pisiferal possesses high cytotoxicity against all investigated cell lines at a concentration between 9.3 ± 0.6 and 14.38 ± 1.4 µM.

Manipulating root water supply elicits major shifts in the shoot proteome.

Substantial reductions in yield caused by drought stress can occur when parts of the root system experience water deficit even though other parts have sufficient access to soil water. To identify proteins associated to drought signaling, rice (Oryza sativa L. cv. IR64.) plants were transplanted into plastic pots with an internal wall dividing each pot into two equal compartments, allowing for equal distribution of soil and the root system between these compartments. The following treatments were applied: either both compartments were watered daily ("wet" roots), or water was withheld from both compartments ("dry" roots), or water was withheld from only one of the two compartments in each pot ("wet" and "dry" roots). The substantial differences in physiological parameters of different growth conditions were accompanied by differential changes in protein abundances. Label-free quantitative shotgun proteomics have resulted in identification of 1383 reproducible proteins across all three conditions. Differentially expressed proteins were categorized within 17 functional groups. The patterns observed were interesting in that in some categories such as protein metabolism and oxidation-reduction, substantial numbers of proteins were most abundant when leaves were receiving signals from "wet" and "dry" roots. In yet other categories such as transport, several key transporters were surprisingly abundant in leaves supported by partially or completely droughted root systems, especially plasma membrane and vacuolar transporters. Stress-related proteins behaved very consistently by increasing in droughted plants but notably some proteins were most abundant when roots of the same plant were growing in both wet and dry soils. Changes in carbohydrate-processing proteins were consistent with the passive accumulation of soluble sugars in shoots under drought, with hydrolysis of sucrose and starch synthesis both enhanced. These results suggest that drought signals are complex interactions and not simply the additive effect of water supply to the roots.

Shotgun proteomic analysis of the Mexican lime tree infected with "CandidatusPhytoplasma aurantifolia".

Infection of Mexican lime trees (Citrus aurantifolia L.) with the specialized bacterium "CandidatusPhytoplasma aurantifolia" causes witches' broom disease. Witches' broom disease has the potential to cause significant economic losses throughout western Asia and North Africa. We used label-free quantitative shotgun proteomics to study changes in the proteome of Mexican lime trees in response to infection by "Ca. Phytoplasma aurantifolia". Of 990 proteins present in five replicates of healthy and infected plants, the abundances of 448 proteins changed significantly in response to phytoplasma infection. Of these, 274 proteins were less abundant in infected plants than in healthy plants, and 174 proteins were more abundant in infected plants than in healthy plants. These 448 proteins were involved in stress response, metabolism, growth and development, signal transduction, photosynthesis, cell cycle, and cell wall organization. Our results suggest that proteomic changes in response to infection by phytoplasmas might support phytoplasma nutrition by promoting alterations in the host's sugar metabolism, cell wall biosynthesis, and expression of defense-related proteins. Regulation of defense-related pathways suggests that defense compounds are induced in interactions with susceptible as well as resistant hosts, with the main differences between the two interactions being the speed and intensity of the response.

Proteomic analysis of rice anthers under salt stress.

Salinity is a major factor that limits rice production worldwide. Rice is considered generally to be sensitive to salt stress during the reproductive stage. To determine the molecular mechanisms of salt tolerance at the reproductive stage, anther proteomic patterns for two contrasting rice genotypes IR64 (salt sensitive) and Cheriviruppu (salt tolerant) under salt stress were compared. Plants were grown in a greenhouse and salt stress (100 mM NaCl) was imposed at the booting stage. Anther samples were collected from control and salt-treated plants at the anthesis stage. The Na(+)/K(+) ratio in IR64 anthers under salt stress was >1.7 times greater than that under control conditions, whereas no significant change was observed in Cheriviruppu. We also observed an 83% reduction in IR64 pollen viability, whereas this reduction was only 23% in Cheriviruppu. Of 454 protein spots detected reproducibly on two-dimensional electrophoresis gels, 38 showed significant changes in at least one genotype in response to stress. Using Mass spectrometry (MALDI TOF/TOF) analysis, we identified 18 protein spots that were involved in several processes that might increase plant adaptation to salt stress, such as carbohydrate/energy metabolism, anther wall remodelling and metabolism, and protein synthesis and assembly. Three isoforms of fructokinase-2 were upregulated only in Cheriviruppu under salt stress. This upregulation might result in increased starch content in pollen, which would support pollen growth and development under salt stress. The results also suggested that anther and pollen wall remodelling/metabolism proteins contribute to the tolerance of rice to salt stress.

Shotgun proteomic analysis of long-distance drought signaling in rice roots.

Rice (Oryza sativa L. cv. IR64) was grown in split-root systems to analyze long-distance drought signaling within root systems. This in turn underpins how root systems in heterogeneous soils adapt to drought. The approach was to compare four root tissues: (1) fully watered; (2) fully droughted and split-root systems where (3) one-half was watered and (4) the other half was droughted. This was specifically aimed at identifying how droughted root tissues altered the proteome of adjacent wet roots by hormone signals and how wet roots reciprocally affected dry roots hydraulically. Quantitative label-free shotgun proteomic analysis of four different root tissues resulted in identification of 1487 nonredundant proteins, with nearly 900 proteins present in triplicate in each treatment. Drought caused surprising changes in expression, most notably in partially droughted roots where 38% of proteins were altered in level compared to adjacent watered roots. Specific functional groups changed consistently in drought. Pathogenesis-related proteins were generally up-regulated in response to drought and heat-shock proteins were totally absent in roots of fully watered plants. Proteins involved in transport and oxidation-reduction reactions were also highly dependent upon drought signals, with the former largely absent in roots receiving a drought signal while oxidation-reduction proteins were strongly present during drought. Finally, two functionally contrasting protein families were compared to validate our approach, showing that nine tubulins were strongly reduced in droughted roots while six chitinases were up-regulated, even when the signal arrived remotely from adjacent droughted roots.

A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat.

Drought is one of the major factors limiting the yield of wheat (Triticum aestivum L.) particularly during grain filling. Under terminal drought condition, remobilization of pre-stored carbohydrates in wheat stem to grain has a major contribution in yield. To determine the molecular mechanism of stem reserve utilization under drought condition, we compared stem proteome patterns of two contrasting wheat landraces (N49 and N14) under a progressive post-anthesis drought stress, during which period N49 peduncle showed remarkably higher stem reserves remobilization efficiency compared to N14. Out of 830 protein spots reproducibly detected and analyzed on two-dimensional electrophoresis gels, 135 spots showed significant changes in at least one landrace. The highest number of differentially expressed proteins was observed in landrace N49 at 20days after anthesis when active remobilization of dry matter was observed, suggesting a possible involvement of these proteins in effective stem reserve remobilization of N49. The identification of 82 of differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in leaf senescence, oxidative stress defense, signal transduction, metabolisms and photosynthesis which might enable N49 to efficiently remobilized its stem reserves compared to N14. The up-regulation of several senescence-associated proteins and breakdown of photosynthetic proteins in N49 might reflect the fact that N49 increased carbon remobilization from the stem to the grains by enhancing senescence. Furthermore, the up-regulation of several oxidative stress defense proteins in N49 might suggest a more effective protection against oxidative stress during senescence in order to protect stem cells from premature cell death. Our results suggest that wheat plant might response to soil drying by efficiently remobilize assimilates from stem to grain through coordinated gene expression.

Cold acclimation proteome analysis reveals close link between the up-regulation of low-temperature associated proteins and vernalization fulfillment.

Low-temperature (LT) stress is one of the major limiting factors in cereal production in cold high-altitude mountainous areas of Iran where cereals are exposed to variable periods of temperatures in the vernalization range during the autumn season. Cereals regulate their development through adaptive mechanisms that are responsive to low but nonfreezing temperatures. We exploited a proteomic approach to determine the interrelationship between vernalization fulfillment and expression of low-temperature (LT)-induced protein in most hardy Norstar and semi-hardy Azar2 wheat (Triticum aestivum L. em. Thell). These cultivars were subjected to 12 h of cold acclimating temperature (2 °C) over a period of 0-89 days. LT tolerance, as measured by LT50, and vernalization fulfillment, as estimated from final leaf number (FLN), was determined at intervals throughout the acclimation period. A significant decrease in FLN associated with LT treatment indicated that Norstar and Azar2 had vernalization responses. Azar2 achieved its vernalization fulfillment and maximum LT tolerance (∼ -8 °C) by 28 days of acclimation. However, Norstar had a longer vernalization requirement (between 35 and 42 days) and reached vernalization fulfillment and maximum LT tolerance (∼ -18.7 °C) about the same time as vernalization fulfillment. We applied a two-dimensional electrophoresis-based proteomics approach to analyze changes in the leaf proteome of two genotypes, Norstar and Azar2, during cold acclimation. Using MALDI-TOF/TOF mass spectrometry, 66 LT-associated proteins could significantly be identified. These proteins were categorized into cold-regulated proteins, antifreezing proteins, oxidative stress defense, photosynthesis, chloroplast post-transcriptional regulation, metabolisms, and protein synthesis. A close association between the vernalization fulfillment and the start of a decline in the protein accumulation of hardy Norstar with a long vernalization requirement and semi-hardy Azar2 with a short vernalization requirement was observed. This finding supported the hypothesis that developmental trait which was regulated by vernalization had a regulatory influence over LT proteome response and highlight a close link between the up-regulation of LT-associated proteins and vernalization fulfillment at the molecular level in wheat.