In this study, phenolic compounds were analyzed in developing berries of four Canadian grown sea buckthorn ('Hippophae rhamnoides' L.) cultivars ('RC-4', 'E6590', 'Chuyskaya' and 'Golden Rain') and in leaves of two of these cultivars. Among phenolic acids, p-coumaric acid was the highest in berries, while gallic acid was predominant in leaves. In the flavonoid class of compounds, myricetin/rutin, kaempferol, quercetin and isorhamnetin were detected in berries and leaves. Berries of the 'RC-4' cultivar had approximately P2-fold higher levels of myricetin and quercetin at 17.5 mg and 17.2 mg/100 g FW, respectively, than the other cultivars. The flavonoid content in leaves was considerably more than in berries with rutin and quercetin levels up to 135 mg and 105 mg/100 g FW, respectively. Orthologs of 15 flavonoid biosynthesis pathway genes were identified within the transcriptome of sea buckthorn mature seeds. Semiquantitative RT-PCR analysis of these genes in developing berries indicated relatively higher expression of genes such as CHS, F30H, DFR and LDOX in the 'RC-4' cultivar than in the 'Chuyskaya' cultivar. Vitamin C levels in ripened berries of the Canadian cultivars were on the high end of the concentration range reported for most other sea buckthorn cultivars. Orthologs of genes involved in vitamins C and E biosynthesis were also identified, expanding the genomic resources for this nutritionally important plant.

Most molecular-genetic studies of plant defense responses to arthropod herbivores have focused on insects. However, plant-feeding mites are also pests of diverse plants, and mites induce different patterns of damage to plant tissues than do well-studied insects (e.g. lepidopteran larvae or aphids). The two-spotted spider mite ('Tetranychus urticae') is among the most significant mite pests in agriculture, feeding on a staggering number of plant hosts. To understand the interactions between spider mite and a plant at the molecular level, we examined reciprocal genome-wide responses of mites and its host Arabidopsis ('Arabidopsis thaliana'). Despite differences in feeding guilds, we found that transcriptional responses of Arabidopsis to mite herbivory resembled those observed for lepidopteran herbivores. Mutant analysis of induced plant defense pathways showed functionally that only a subset of induced programs, including jasmonic acid signaling and biosynthesis of indole glucosinolates, are central to Arabidopsis's defense to mite herbivory. On the herbivore side, indole glucosinolates dramatically increased mite mortality and development times. We identified an indole glucosinolate dose-dependent increase in the number of differentially expressed mite genes belonging to pathways associated with detoxification of xenobiotics. This demonstrates that spider mite is sensitive to Arabidopsis defenses that have also been associated with the deterrence of insect herbivores that are very distantly related to chelicerates. Our findings provide molecular insights into the nature of, and response to, herbivory for a representative of a major class of arthropod herbivores.

As a resource allocation strategy, plant growth and defense responses are generally mutually antagonistic. Brassinosteroid (BR) regulates many aspects of plant development and stress responses, however, genetic evidence of its integrated effects on plant growth and stress tolerance is lacking. We overexpressed the 'Arabidopsis' BR biosynthetic gene 'AtDWF4' in the oilseed plant 'Brassica napus' and scored growth and stress response phenotypes. The transgenic 'B. napus' plants, in comparison to wild type, displayed increased seed yield leading to increased overall oil content per plant, higher root biomass and root length, significantly better tolerance to dehydration and heat stress, and enhanced resistance to necrotrophic fungal pathogens 'Leptosphaeria maculans' and 'Sclerotinia sclerotiorum'. Transcriptome analysis supported the integrated effects of BR on growth and stress responses; in addition to BR responses associated with growth, a predominant plant defense signature, likely mediated by BES1/BZR1, was evident in the transgenic plants. These results establish that BR can interactively and simultaneously enhance abiotic and biotic stress tolerance and plant productivity. The ability to confer pleiotropic beneficial effects that are associated with different agronomic traits suggests that BR-related genes may be important targets for simultaneously increasing plant productivity and performance under stress conditions.

Brassinosteroids (BRs) are essential for proper plant growth and development and also protect plants from a variety of environmental stresses. Seeds contain relatively high levels of BRs, and BRs have been implicated in embryonic patterning and germination. How BR levels in seeds impact germination, growth, and stress tolerance in early seedlings is currently not known. To assess this, the BR biosynthetic gene 'AtDWF4' was overexpressed in 'Arabidopsis' under the control of a seed-specific oleosin promoter. The resulting transgenic seedlings could overcome inhibition of germination caused by exogenous abscisic acid (ABA) and the seedlings were more tolerant to cold stress compared to wild-type and vector control seedlings. Transcript levels of 'COR15A', a cold-responsive gene with an established function in cold tolerance, were approximately twofold higher in transgenic seedlings than in control seedlings under cold conditions. These results establish a role for BRs in opposing the inhibitory effects of ABA in seed germination and in promoting cold stress tolerance in early 'Arabidopsis' seedlings.

Brassinosteroids are a newly reported class of plant growth phytohormones found in plants throughout the plant kingdom. Functioning at very low concentrations, they play an essential role in improving biomass yield and stress tolerance. There are no reports in the literature of the genetic variability of responsiveness of brassinosteroids in wheat; most studies on brassinosteroids have focused on the physiological eVects of exogenous addition of brassinosteroids. Our aim was to study the genetic variation in the responsiveness of a doubled haploid durum wheat population to three brassinosteroid concentrations using the leaf unrolling test, which is a simple bioassay to test brassinosteroid activity. An F1-derived doubled haploid population of 77 individuals from the cross StrongWeld/Blackbird was used to construct a genetic map of 427 molecular marker loci. The leaf unrolling test was performed on the parents and doubled haploid genotypes of the population using 0.2, 2 and 20 nM brassinosteroid concentrations. The results indicated significant diVerences in leaf unrolling between the two parents, doubled haploid genotypes, treatments and genotype-by-treatment combinations. Transgressive segregation beyond StrongWeld of leaf unrolling was observed for all concentrations, with the strongest response at 20 nM. Putative quantitative trait loci were revealed in the intervals 'Xgwm2–Xbarc45' on chromosome 3A and 'Xwmc643a–Xwmc625a' on chromosome 3B. Additional quantitative trait loci were associated with markers 'Xwmc48a', 'Xwmc511', 'Xwmc89a' and 'Xgwmc692' on chromosome 4B, and 'Xwmc17' on chromosome 7A. This work should enhance the understanding of the relationship between stress tolerance and productivity, and responsiveness to brassinosteroids.

Background: Brassinosteroids (BRs) play crucial roles in plant development and also promote tolerance to a range of abiotic stresses. Although much has been learned about their roles in plant development, the mechanisms by which BRs control plant stress responses and regulate stress-responsive gene expression are not fully known. Since BR interacts with other plant hormones, it is likely that the stress tolerance conferring ability of BR lies in part in its interactions with other stress hormones. Results: Using a collection of Arabidopsis mutants that are either deficient in or insensitive to abscisic acid (ABA), ethylene (ET), jasmonic acid (JA) and salicylic acid (SA), we studied the effects of 24-epibrassinloide (EBR) on basic thermotolerance and salt tolerance of these mutants. The positive impact of EBR on thermotolerance in proportion to wild type was evident in all mutants studied, with the exception of the SA-insensitive 'npr1-1' mutant. EBR could rescue the ET-insensitive 'ein2' mutant from its hypersensitivity to salt stress-induced inhibition of seed germination, but remained ineffective in increasing the survival of 'eto1-1' (ET-overproducer) and 'npr1-1' seedlings on salt. The positive effect of EBR was significantly greater in the ABA-deficient 'aba1-1' mutant as compared to wild type, indicating that ABA masks BR effects in plant stress responses. Treatment with EBR increased expression of various hormone marker genes in both wild type and mutant seedlings, although to different levels. Conclusions: These results together indicate that the redox-sensitive protein NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1), a master regulator of SA-mediated defense genes, is likely a critical component of EBR-mediated increase in thermotolerance and salt tolerance, but it is not required for EBR-mediated induction of 'PR-1' ('PATHOGENESIS-RELATED1') gene expression; that BR exerts anti-stress effects independently as well as through interactions with other hormones; that ABA inhibits BR effects during stress; and that BR shares transcriptional targets with other hormones.

The small acidic protein p23 is best described as a co-chaperone of Hsp90, an essential molecular chaperone in eukaryotes. p23 binds to the ATP-bound form of Hsp90 and stabilizes the Hsp90-client protein complex by slowing down ATP turnover. The stabilizing activity of p23 was first characterized in studies of steroid receptor-Hsp90 complexes. Earlier studies of the Hsp90 chaperone complex in plants suggested that a p23-like stabilizing activity was absent in plant cell lysates. Here, we show that p23-like proteins are present in plants and are capable of binding Hsp90, but unlike human p23 and yeast ortholog Sba1, the plant p23-like proteins do not stabilize the steroid receptor-Hsp90 complexes formed in wheat germ lysate. Furthermore, these proteins do not inhibit the ATPase activity of plant Hsp90. While transcripts of 'Arabidopsis thaliana p23-1' and 'Atp23-2' were detected under normal growing conditions, those of the closely related 'Brassica napus p23-1' were present only after moderate heat stress. These observations suggest that p23-like proteins in plants are conserved in their binding to Hsp90 but have evolved mechanisms of action different from their yeast and animal counterparts.

The essential eukaryotic molecular chaperone Hsp90 operates with the help of different co-chaperones, which regulate its ATPase activity and serve as adaptors to recruit client proteins and other molecular chaperones, such as Hsp70, to the Hsp90 complex. Several Hsp90 and Hsp70 co-chaperones contain the tetratricopeptide repeat (TPR) domain, which interacts with the highly conserved EEVD motif at the C-terminal ends of Hsp90 and Hsp70. The acidic side chains in EEVD interact with a subset of basic residues in the TPR binding pocket called a 'carboxylate clamp'. Since the carboxylate clamp residues are conserved in the TPR domains of known Hsp90/Hsp70 co-chaperones, we carried out an in silico search for TPR proteins in 'Arabidopsis' and rice comprising of at least one three-motif TPR domain with conserved amino acid residues required for Hsp90/Hsp70 binding. This approach identified in Arabidopsis a total of 36 carboxylate clamp (CC)-TPR proteins, including 24 novel proteins, with potential to interact with Hsp90/Hsp70. The newly identified CC-TPR proteins in 'Arabidopsis' and rice contain additional protein domains such as ankyrin, SET, octicosapeptide/Phox/Bem1p (Phox/PB1), DnaJ-like, thioredoxin, FBD and F-box, and protein kinase and U-box, indicating varied functions for these proteins. To provide proof-of-concept of the newly identified CC-TPR proteins for interaction with Hsp90, we demonstrated interaction of AtTPR1 and AtTPR2 with AtHsp90 in yeast two-hybrid and 'in vitro' pull down assays. These findings indicate that the 'in silico' approach used here successfully identified in a genome-wide context CC-TPR proteins with potential to interact with Hsp90/Hsp70, and further suggest that the Hsp90/Hsp70 system relies on TPR co-chaperones more than it was previously realized.

Background: Sea buckthorn ('Hippophae rhamnoides' L.) is a hardy, fruit-producing plant known historically for its medicinal and nutraceutical properties. The most recognized product of sea buckthorn is its fruit oil, composed of seed oil that is rich in essential fatty acids, linoleic (18:2ω-6) and α-linolenic (18:3ω-3) acids, and pulp oil that contains high levels of monounsaturated palmitoleic acid (16:1ω-7). Sea buckthorn is fast gaining popularity as a source of functional food and nutraceuticals, but currently has few genomic resources; therefore, we explored the fatty acid composition of Canadiangrown cultivars (ssp. 'mongolica') and the sea buckthorn seed transcriptome using the 454 GS FLX sequencing technology. Results: GC-MS profiling of fatty acids in seeds and pulp of berries indicated that the seed oil contained linoleic and α-linolenic acids at 33-36% and 30-36%, respectively, while the pulp oil contained palmitoleic acid at 32-42%. 454 sequencing of sea buckthorn cDNA collections from mature seeds yielded 500,392 sequence reads, which identified 89,141 putative unigenes represented by 37,482 contigs and 51,659 singletons. Functional annotation by Gene Ontology and computational prediction of metabolic pathways indicated that primary metabolism (protein.nucleic acid.carbohydrate. lipid) and fatty acid and lipid biosynthesis pathways were highly represented categories. Sea buckthorn sequences related to fatty acid biosynthesis genes in Arabidopsis were identified, and a subset of these was examined for transcript expression at four developing stages of the berry. Conclusion: This study provides the first comprehensive genomic resources represented by expressed sequences for sea buckthorn, and demonstrates that the seed oil of Canadian-grown sea buckthorn cultivars contains high levels of linoleic acid and α-linolenic acid in a close to 1:1 ratio, which is beneficial for human health. These data provide the foundation for further studies on sea buckthorn oil, the enzymes involved in its biosynthesis, and the genes involved in the general hardiness of sea buckthorn against environmental conditions.

The plant hormone brassinosteroid (BR) plays essential roles in plant growth and development, while also controlling plant stress responses. This dual ability of BR is intriguing from a echanistic point of view and as a viable solution for stabilizing crop yields under the changing limatic conditions. Here we report a time course analysis of BR responses under both stress and no-stress conditions, the results of which establish that BR incorporates many stress-related features even under no-stress conditions, which are then accompanied by a dynamic stress response under unfavourable conditions. Found within the BR transcriptome were distinct molecular signatures of two stress hormones, abscisic acid and jasmonic acid, which were correlated with enhanced endogenous levels of the two hormones in BR-treated seedlings. The marked presence of genes related to protein metabolism and modification, defence responses and calcium signalling highlights the significance of their associated mechanisms and roles in BR processes. Functional analysis of loss-of-function mutants of a subset of genes selected from the BR transcriptome identified abiotic stress-related roles for 'ACID PHOSPHATASE5 (ACP5)', 'WRKY33', JACALIN-RELATED LECTIN1-3' ('JAC-LEC1-3') and a 'BR-RESPONSIVE-RECEPTOR-LIKE KINASE' ('BRRLK'). Overall, the results of this study provide a clear link between the molecular changes impacted by BR and its ability to confer broad-range stress tolerance, emphasize the importance of post-translational modification and protein turnover as BR regulatory mechanisms and demonstrate the BR transcriptome as a repertoire of new stress-related regulatory and structural genes.