Metabolite profiling and expression analysis of flavonoid, vitamin C and tocopherol biosynthesis genes in the antioxidant-rich sea buckthorn ('Hippophae rhamnoides' L.)
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.
Overexpression of the Brassinosteroid Biosynthetic Gene 'AtDWF4' in 'Arabidopsis' Seeds Overcomes Abscisic Acid-induced Inhibition of Germination and Increases Cold Tolerance in Transgenic Seedlings
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.
Brassinosteroid: a biotechnological target for enhancing crop yield and stress tolerance
Brassinosteroids (BRs) are a group of naturally occurring plant steroidal compounds with wide ranging biological activity. Because BRs control several important agronomic traits such as flowering time, plant architecture, seed yield and stress tolerance, the genetic manipulation of BR biosynthesis, conversion or perception offers a unique possibility of significantly increasing crop yields through both changing plant metabolism and protecting plants from environmental stresses. Genetic manipulation of BR activity has indeed led to increases in crop yield by 20–60%, confirming the value of further research on BRs to improve productivity.
Characterization of plant p23-like proteins for their co-chaperone activities
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.
Reciprocal Responses in the Interaction between Arabidopsis and the Cell-Content-Feeding Chelicerate Herbivore Spider Mite
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.
Brassinosteroids Confer Stress Tolerance
Brassinosteroids (BRs) are a group of plant steroidal hormones that are structurally related to animal and insect steroid hormones. BRs regulate a wide range of physiological responses in plants, including cell elongation, photomorphogenesis, xylem differentiation, seed germination, and stress responses. Although the growth-promoting properties of BRs were recognized in the early 1970s, the first genetic evidence to suggest that BRs are essential for proper plant development came with the isolation of the BR-deficient mutants det2 (de-etiolated2) and cpd (constitutive photomorphogenic dwarf). Isolation and sequence analysis of DET2 and CPD genes revealed that the encoded proteins share sequence similarities with steroid 5a-reductases and steroid hydroxylases, respectively, indicating a role for these proteins in steroid metabolism. Indeed, feeding det2 and cpd mutant seedlings with BRs rescued their mutant phenotypes to wild-type in a dose-dependent manner, clearly establishing the roles of DET2 and CPD in BR biosynthesis. Numerous other Arabidopsis BR-deficient and BR-insensitive mutants, displaying phenotypic alterations such as dwarfism, small dark-green leaves, a compact rosette structure, delayed flowering and senescence, and reduced fertility, were instrumental in the identification of BR signaling components and in understanding to some extent how BR regulates gene expression. Numerous reviews detailing BR effects on plant growth and development and BR signaling mechanisms have surfaced in the recent literature; these aspects have therefore been discussed only briefly here. The present chapter is focused on the relatively less explored topic of BR-mediated stress responses in plants and highlights the progress made towards understanding the molecular basis of BRmediated plant stress tolerance.
Brassinosteroid-mediated stress tolerance in Arabidopsis shows interactions with abscisic acid, ethylene and salicylic acid pathways
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.
Overexpression of the brassinosteroid biosynthetic gene 'DWF4' in 'Brassica napus' simultaneously increases seed yield and stress tolerance
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.
Brassinosteroid leaf unrolling QTL mapping in durum wheat
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.
Identification of Differentially Expressed Genes in Brassinosteroid-Treated 'Brassica napus' Seedlings
Brassinosteroid-mediated gene expression changes have been reported primarily in 'Arabidopsis thaliana' but not in its close relative Brassica napus. To obtain an initial idea of the molecular changes induced by long-term exposure to 24-epibrassinolide (EBR) in 'B. napus' seedlings, we used the differential display-reverse transcription PCR technique. Six differentially expressed cDNAs were isolated and characterized. These encode a mitochondrial transcription termination factor (mTERF)-related protein, glycine-rich protein 22 (GRP22), myrosinase, 3-ketoacyl-CoA thiolase, and a copia-like polyprotein. The first four were upregulated in EBR-treated seedlings while the latter was expressed at higher levels in untreated seedlings. Transcripts of mTERF-related protein, GRP22, and myrosinase were present at higher levels in treated seedlings under nonstress conditions, whereas those of 3-ketoacyl-CoA thiolase rose to higher levels in treated seedlings during exposure to heat stress. The results of the present study indicate that EBR treatment in 'B. napus' leads to substantial changes in the expression levels of genes involved in a variety of physiologic responses. The results provide a useful framework for further research into EBR-mediated molecular changes in 'B. napus', which will also add to our understanding of how brassinosteroids mediate stress tolerance in this agriculturally important oil crop.