Adaptive Evolution of Chalcone Isomerase Superfamily in Fagaceae.

Chalcone Isomerase (CHI) catalyzes the biosynthesis of flavonoids and secondary metabolism in plants. Currently, there is no systematic analysis of CHIs gene family in Fagaceae which is available. In this study, twenty-two CHI proteins were identified in five species of the Fagaceae family. The CHI superfamily in Fagaceae can be classified into three subfamilies and five groups using phylogenetic analysis, analysis of physicochemical properties, and structural prediction. Results indicated that serine (Ser) and isoleucine (Ile) residues determine the substrate preferred by active Type I Fagaceae CHI, and the chalcone isomerase-like (CHIL) of Fagaceae had active site residues. Adaptive analysis of CHIs showed that CHIs are subject to selection pressure. The active CHI gene of Fagaceae was located in the cytoplasm, and it had the typical gene structure of CHI and contains four exons. All the twenty-two identified CHIs had the conserved domain motif 3, and the different groups had their own structural characteristics. In the process of fatty acid binding protein (FAP) evolution to CHIL and CHI, the physical and chemical properties of proteins also had significant differences in addition to changes in protein functions.

Identification and expression analysis of starch branching enzymes involved in starch synthesis during the development of chestnut (Castanea mollissima Blume) cotyledons.

Chinese chestnut (Castanea mollissima Blume) is native to China and distributes widely in arid and semi-arid mountain area with barren soil. As a perennial crop, chestnut is an alternative food source and acts as an important commercial nut tree in China. Starch is the major metabolite in nuts, accounting for 46 ~ 64% of the chestnut dry weight. The accumulation of total starch and amylopectin showed a similar increasing trend during the development of nut. Amylopectin contributed up to 76% of the total starch content at 80 days after pollination (DAP). The increase of total starch mainly results from amylopectin synthesis. Among genes associated with starch biosynthesis, CmSBEs (starch branching enzyme) showed significant increase during nut development. Two starch branching enzyme isoforms, CmSBE I and CmSBE II, were identified from chestnut cotyledon using zymogram analysis. CmSBE I and CmSBE II showed similar patterns of expression during nut development. The accumulations of CmSBE transcripts and proteins in developing cotyledons were characterized. The expressions of two CmSBE genes increased from 64 DAP and reached the highest levels at 77 DAP, and SBE activity reached its peak at 74 DAP. These results suggested that the CmSBE enzymes mainly contributed to amylopectin synthesis and influenced the amylopectin content in the developing cotyledon, which would be beneficial to chestnut germplasm selection and breeding.

Salicylic acid inhibits enzymatic browning of fresh-cut Chinese chestnut (Castanea mollissima) by competitively inhibiting polyphenol oxidase.

The inhibitory effect and associated mechanisms of salicylic acid (SA) on the browning of fresh-cut Chinese chestnut were investigated. Shelled and sliced chestnuts were immersed in different concentrations of an SA solution, and the browning of the chestnut surface and interior were inhibited. The activities of polyphenol oxidase (PPO) and peroxidase (POD) extracted from chestnuts were measured in the presence and absence of SA. SA at concentrations higher than 0.3g/L delayed chestnut browning by significantly inhibiting the PPO activity (P<0.01), and the POD activity was not significantly affected (P>0.05). The binding and inhibition modes of SA with PPO and POD, determined by AUTODOCK 4.2 and Lineweaver-Burk plots, respectively, established SA as a competitive inhibitor of PPO.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Jasmonate biosynthesis in legume and actinorhizal nodules.

Jasmonic acid (JA) is a plant signalling compound that has been implicated in the regulation of mutualistic symbioses. In order to understand the spatial distribution of JA biosynthetic capacity in nodules of two actinorhizal species, Casaurina glauca and Datisca glomerata, and one legume, Medicago truncatula, we determined the localization of allene oxide cyclase (AOC) which catalyses a committed step in JA biosynthesis. In all nodule types analysed, AOC was detected exclusively in uninfected cells. The levels of JA were compared in the roots and nodules of the three plant species. The nodules and noninoculated roots of the two actinorhizal species, and the root systems of M. truncatula, noninoculated or nodulated with wild-type Sinorhizobium meliloti or with mutants unable to fix nitrogen, did not show significant differences in JA levels. However, JA levels in all plant organs examined increased significantly on mechanical disturbance. To study whether JA played a regulatory role in the nodules of M. truncatula, composite plants containing roots expressing an MtAOC1-sense or MtAOC1-RNAi construct were inoculated with S. meliloti. Neither an increase nor reduction in AOC levels resulted in altered nodule formation. These data suggest that jasmonates are not involved in the development and function of root nodules.

Salicylic acid induces H2O2 production and endochitinase gene expression but not ethylene biosynthesis in Castanea sativa in vitro model system.

Salicylic acid (SA), ethylene (ET), and wounding are all known to influence plant defense response. Experiments attempting to determine SA's relation to ET biosynthesis and defense gene expression have shown conflicting results. To confront this, we developed an in vitro model system to investigate how SA affects ET biosynthesis, hydrogen peroxide (H(2)O(2)) production and endochitinase gene expression in the European chestnut. ET measurements of in vitro shoots indicated a critical time point for SA exogenous application, enabling us to study its effects independent of ET. In addition, ET measurements demonstrated that its own increased biosynthesis was a response to wounding but not to SA treatment. Application of the ET biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), on wounded and SA-treated shoots blocked wounding-induced ET production. Interestingly, SA inhibited ET production, but to a lesser extent than AVG. Additionally, SA also induced the accumulation of endochitinase transcript level. Likewise, a sensitive tissue-print assay showed that SA further increased the level of H(2)O(2). Yet, SA-induced endochitinase gene expression and SA-enhanced H(2)O(2) production levels were independent of ET. The cumulative results indicate that SA acts as an inducer of endochitinase PR gene expression and of H(2)O(2) oxidative burst. This suggests that SA is a component of the signal transduction pathway leading to defense against pathogens in chestnut. Further, the model system developed for this experiment should facilitate the deciphering of defense signaling pathways and their cross-talk. Moreover, it should also benefit the study of trees of long generation time that are known to be recalcitrant to in vitro studies.

Genetic diversity in Nothofagus alessandrii (Fagaceae), an endangered endemic tree species of the coastal maulino forest of Central Chile.

BACKGROUND AND AIMS: The endemic tree Nothofagus alessandrii (Fagaceae) has been historically restricted to the coastal range of Region VII of central Chile, and its forests have been increasingly destroyed and fragmented since the end of the 19th century. In this study, the patterns of within- and among-population genetic diversity in seven fragments of this endangered narrowly endemic tree were examined. METHODS: Allozyme electrophoresis of seven loci of N. alessandrii was used to estimate genetic diversity, genetic structure and gene flow. KEY RESULTS: High levels of genetic diversity were found as shown by mean expected heterozygosity (H(e) = 0.182 +/- 0.034), percentage of polymorphic loci (P(p) = 61.2 %), mean number of alleles per locus (A = 1.8) and mean number of alleles per polymorphic locus (A(p) = 2.3). Genetic differentiation was also high (G(ST) = 0.257 and Nm = 0.7). These values are high compared with more widespread congeneric species. CONCLUSIONS: Despite its endemic status and restricted geographical range N. alessandrii showed high levels of genetic diversity. The observed patterns of diversity are explained in part by historical processes and more recent human fragmentation.

Glutathione transferase, but not agglutinin, is a dormancy-related protein in Castanea crenata trees.

The annual changes in Japanese chestnut (Castanea crenata Sieb. et Zucc.) agglutinin (CCA) were investigated by both protein and RNA blotting analyses, to clarify whether CCA has a function as storage protein. In the woody part of shoots and leaves, CCA expression was only detected at both the protein and RNA levels in May and June. In buds, the CCA protein and mRNA expressions were both restricted to April. However, the amount of accumulated CCA was too low to act as a nitrogen reserve. No expression was observed in the bark at any time point, suggesting that bark does not contain either CCA or CCA-like proteins. These results suggest that CCA may be required in young organs as a defense protein, rather than as a storage protein. In addition, CCA was not related to dormancy, unlike some other woody plant bark lectins. In contrast to CCA, a 28kDa polypeptide was observed to accumulate during dormancy. Sequence analysis indicated that this polypeptide was a glutathione transferase. After cDNA cloning, RNA blot analyses indicated that this glutathione transferase was strongly expressed in woody parts during mid-winter. In shoots, this protein represented approximately 10% of the total soluble protein content. Therefore, in Japanese chestnut trees, glutathione transferase may play a nitrogen storage role in addition to its intrinsic defensive role against stresses during dormancy.