RESUMEN

Water use efficiency (WUE) is crucial for apple tree fitness and survival, especially in response to climatic changes. The receptor-like kinase FERONIA is reportedly an essential regulator of plant stress responses, but its role in regulating WUE under water deficit conditions is unclear. Here, we found that overexpressing the apple FERONIA receptor kinase gene, MdMRLK2, enhanced apple WUE under long-term water deficit conditions. Under drought treatment, 35S::MdMRLK2 apple plants exhibited higher photosynthetic capacity and antioxidant enzyme activities than wild-type (WT) plants. 35S::MdMRLK2 apple plants also showed increased biomass accumulation, root activity, and water potential compared to WT plants. Moreover, MdMRLK2 physically interacts with and phosphorylates cinnamoyl-CoA reductase 1, MdCCR1, an enzyme essential for lignin synthesis, at position Ser260. This interaction likely contributed to increased vessel density, vascular cylinder area, and lignin content in 35S::MdMRLK2 apple plants under drought conditions. Therefore, our findings reveal a novel function of MdMRLK2 in regulating apple WUE under water deficit conditions.

RESUMEN

MAIN CONCLUSION: The identification of a functional cinnamoyl-CoA reductase enzyme from Cinnamomum cassia involved in trans-cinnamaldehyde biosynthesis offers the potential for enhancing trans-cinnamaldehyde production through genetic engineering. A significant accumulation of trans-cinnamaldehyde has been found in the bark tissues of C. cassia, used in traditional Chinese medicine. trans-Cinnamaldehyde exhibits various pharmacological properties such as anti-inflammatory, analgesic, and protection of the stomach and the digestive tract. However, further elucidation and characterization of the biosynthetic pathway for trans-cinnamaldehyde is required. In this study, we conducted an integrated analysis of trans-cinnamaldehyde accumulation profiles and transcriptomic data from five different C. cassia tissues to identify the genes involved in its biosynthesis. The transcriptome data we obtained included nearly all genes associated with the trans-cinnamaldehyde pathway, with the majority demonstrating high abundance in branch barks and trunk barks. We successfully cloned four C. cassia cinnamoyl-CoA reductases (CcCCRs), a key gene in trans-cinnamaldehyde biosynthesis. We found that the recombinant CcCCR1 protein was the only one that more efficiently converted cinnamoyl-CoA into trans-cinnamaldehyde. CcCCR1 exhibited approximately 14.7-fold higher catalytic efficiency (kcat/Km) compared to the Arabidopsis thaliana cinnamoyl-CoA reductase 1 (AtCCR1); therefore, it can be utilized for engineering higher trans-cinnamaldehyde production as previously reported. Molecular docking studies and mutagenesis experiments also validated the superior catalytic activity of CcCCR1 compared to AtCCR1. These findings provide valuable insights for the functional characterization of enzyme-coding genes and hold potential for future engineering of trans-cinnamaldehyde biosynthetic pathways.

Asunto(s)

RESUMEN

Eugenol, the main component of essential oil from the Syzygium aromaticum clove tree, has great potential as an alternative bioresource feedstock for biosynthesis purposes. Although eugenol degradation to ferulic acid was investigated, an efficient method for directly converting eugenol to targeted natural products has not been established. Herein we identified the inherent inhibitions by simply combining the previously reported ferulic acid biosynthetic pathway and vanillin biosynthetic pathway. To overcome this, we developed a novel biosynthetic pathway for converting eugenol into vanillin, by introducing cinnamoyl-CoA reductase (CCR), which catalyzes conversion of coniferyl aldehyde to feruloyl-CoA. This approach bypasses the need for two catalysts, namely coniferyl aldehyde dehydrogenase and feruloyl-CoA synthetase, thereby eliminating inhibition while simplifying the pathway. To further improve efficiency, we enhanced CCR catalytic efficiency via directed evolution and leveraged an artificialvanillin biosensor for high-throughput screening. Switching the cofactor preference of CCR from NADP+ to NAD+ significantly improved pathway efficiency. This newly designed pathway provides an alternative strategy for efficiently biosynthesizing feruloyl-CoA-derived natural products using eugenol.

Asunto(s)

RESUMEN

Cassava (Manihot esculenta Crantz) is a food and industrial storage root crop with substantial potential to contribute to managing risk associated with climate change due to its inherent resilience and in providing a biodegradable option in manufacturing. In Africa, cassava production is challenged by two viral diseases, cassava brown streak disease (CBSD) and cassava mosaic disease. Here we detect quantitative trait loci (QTL) associated with CBSD in a biparental mapping population of a Tanzanian landrace, Nachinyaya and AR37-80, phenotyped in two locations over three years. The purpose was to use the information to ultimately facilitate either marker-assisted selection or adjust weightings in genomic selection to increase the efficiency of breeding. Results from this study were considered in relation to those from four other biparental populations, of similar genetic backgrounds, that were phenotyped and genotyped simultaneously. Further, we investigated the co-localization of QTL for CBSD resistance across populations and the genetic relationships of parents based on whole genome sequence information. Two QTL on chromosome 4 for resistance to CBSD foliar symptoms and one on each of chromosomes 11 and 18 for root necrosis were of interest. Of significance within the candidate genes underlying the QTL on chromosome 4 are Phenylalanine ammonia-lyase (PAL) and Cinnamoyl-CoA reductase (CCR) genes and three PEPR1-related kinases associated with the lignin pathway. In addition, a CCR gene was also underlying the root necrosis-resistant QTL on chromosome 11. Upregulation of key genes in the cassava lignification pathway from an earlier transcriptome study, including PAL and CCR, in a CBSD-resistant landrace compared to a susceptible landrace suggests a higher level of basal lignin deposition in the CBSD-resistant landrace. Earlier RNAscope® in situ hybridisation imaging experiments demonstrate that cassava brown streak virus (CBSV) is restricted to phloem vessels in CBSV-resistant varieties, and phloem unloading for replication in mesophyll cells is prevented. The results provide evidence for the involvement of the lignin pathway. In addition, five eukaryotic initiation factor (eIF) genes associated with plant virus resistance were found within the priority QTL regions.

RESUMEN

Asarum sieboldii Miq., a perennial herb of the family Aristolochiaceae, is widely used in China to treat cold, fever, aphthous stomatitis, toothache, gingivitis, and rheumatoid arthritis. Methyleugenol is the most representative pharmacological constituent of this medicinal herb. Cinnamoyl-CoA reductase (CCR), which has been well known for occupying a critical position in the lignin biosynthesis pathway, is also shared with the biosynthesis of methyleugenol. To better understand the regulatory mechanisms of methyleugenol biosynthesis, a 1530-bp long promoter region of the AsCCR1 gene was isolated. PLACE and PlantCARE analysis affirmed the existence of the core promoter elements such as TATA and CAAT boxes, abiotic stress-responsive cis-regulation elements like abscisic acid-responsive element, G-box, and MBS in the isolated sequence. The histochemical assay suggested that it was a constitutive promoter, highly expressed in the root tissue. Moreover, the region of -200 bp to ATG (start codon) was enough to drive the expression of It GUS gene. Treatments with low temperature and high concentration of gibberellin or abscisic acid demonstrated the abiotic stress-induced expression of the AsCCR1 promoter. Overall, this study revealed the isolation and characterization of the AsCCR1 promoter. Moreover, it also provided a candidate gene for molecular breeding in A. sieboldii to enhance its pharmacological potential.

Asunto(s)

RESUMEN

Cinnamoyl-CoA reductase (CCR) is a pivotal enzyme in plant lignin synthesis, which has a role in plant secondary cell wall development and environmental stress defense. Alfalfa is a predominant legume forage with excellent quality, but the lignin content negatively affects fodder digestibility. Currently, there is limited information on CCR characteristics, gene expression, and its role in lignin metabolism in alfalfa. In this study, we identified 30 members in the CCR gene family of Medicago sativa. In addition, gene structure, conserved motif, and evolution analysis suggested MsCCR1-7 presumably functioned as CCR, while the 23 MsCCR-likes fell into three categories. The expression patterns of MsCCRs/MsCCR-likes suggested their role in plant development, response to environmental stresses, and phytohormone treatment. These results were consistent with the cis-elements in their promoters. Histochemical staining showed that lignin accumulation gradually deepened with the development, which was consistent with gene expression results. Furthermore, recombinant MsCCR1 and MsCCR-like1 were purified and the kinetic parameters were tested under four substrates. In addition, three-dimensional structure models of MsCCR1 and MsCCR-like1 proteins showed the difference in the substrate-binding motif H212(X)2K215R263. These results will be useful for further application for legume forage quality modification and biofuels industry engineering in the future.

Asunto(s)

RESUMEN

Introduction: Wood formation is closely related to lignin biosynthesis. Cinnamoyl-CoA reductase (CCR) catalyzes the conversion of cinnamoyl-CoA to cinnamaldehydes, which is the initiation of the lignin biosynthesis pathway and a crucial point in the manipulation of associated traits. Liriodendron chinense is an economically significant timber tree. Nevertheless, the underlying mechanism of wood formation in it remains unknown; even the number of LcCCR family members in this species is unclear. Materials and Results: This study aimed to perform a genome-wide identification of genes(s) involved in lignin biosynthesis in L. chinense via RT-qPCR assays and functional verification. Altogether, 13 LcCCR genes were identified that were divided into four major groups based on structural and phylogenetic features. The gene structures and motif compositions were strongly conserved between members of the same groups. Subsequently, the expression patterns analysis based on RNA-seq data indicated that LcCCR5/7/10/12/13 had high expression in the developing xylem at the stem (DXS). Furthermore, the RT-qPCR assays showed that LcCCR13 had the highest expression in the stem as compared to other tissues. Moreover, the overexpression of the LcCCR13 in transgenic tobacco plants caused an improvement in the CCR activity and lignin content, indicating that it plays a key role in lignin biosynthesis in the stems. Discussion: Our research lays a foundation for deeper investigation of the lignin synthesis and uncovers the genetic basis of wood formation in L. chinense.

RESUMEN

Introduction: Lignin is a complex aromatic polymer plays major biological roles in maintaining the structure of plants and in defending them against biotic and abiotic stresses. Cinnamoyl-CoA reductase (CCR) is the first enzyme in the lignin-specific biosynthetic pathway, catalyzing the conversion of hydroxycinnamoyl-CoA into hydroxy cinnamaldehyde. Dalbergia odorifera T. Chen is a rare rosewood species for furniture, crafts and medicine. However, the CCR family genes in D. odorifera have not been identified, and their function in lignin biosynthesis remain uncertain. Methods and Results: Here, a total of 24 genes, with their complete domains were identified. Detailed sequence characterization and multiple sequence alignment revealed that the DoCCR protein sequences were relatively conserved. They were divided into three subfamilies and were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that seven DoCCRs were grouped together with functionally characterized CCRs of dicotyledons involved in developmental lignification. Synteny analysis showed that segmental and tandem duplications were crucial in the expansion of CCR family in D. odorifera, and purifying selection emerged as the main force driving these genes evolution. Cis-acting elements in the putative promoter regions of DoCCRs were mainly associated with stress, light, hormones, and growth/development. Further, analysis of expression profiles from the RNA-seq data showed distinct expression patterns of DoCCRs among different tissues and organs, as well as in response to stem wounding. Additionally, 74 simple sequence repeats (SSRs) were identified within 19 DoCCRs, located in the intron or untranslated regions (UTRs), and mononucleotide predominated. A pair of primers with high polymorphism and good interspecific generality was successfully developed from these SSRs, and 7 alleles were amplified in 105 wild D. odorifera trees from 17 areas covering its whole native distribution. Discussion: Overall, this study provides a basis for further functional dissection of CCR gene families, as well as breeding improvement for wood properties and stress resistance in D. odorifera.

RESUMEN

Sclerotinia sclerotiorum causes severe yield and economic losses for many crop and vegetable species, especially Brassica napus. To date, no immune B. napus germplasm has been identified, giving rise to a major challenge in the breeding of Sclerotinia resistance. In the present study, we found that, compared with a Sclerotinia-susceptible line (J902), a Sclerotinia-resistant line (J964) exhibited better xylem development and a higher lignin content in the stems, which may limit the invasion and spread of S. sclerotiorum during the early infection period. In addition, genes involved in lignin biosynthesis were induced under S. sclerotiorum infection in both lines, indicating that lignin was deposited proactively in infected tissues. We then overexpressed BnaC.CCR2.b, which encodes the first rate-limiting enzyme (cinnamoyl-CoA reductase) that catalyzes the reaction of lignin-specific pathways, and found that overexpression of BnaC.CCR2.b increased the lignin content in the stems of B. napus by 2.28-2.76% under normal growth conditions. We further evaluated the Sclerotinia resistance of BnaC.CCR2.b overexpression lines at the flower-termination stage and found that the disease lesions on the stems of plants in the T2 and T3 generations decreased by 12.2-33.7% and 32.5-37.3% compared to non-transgenic control plants, respectively, at 7days post-inoculation (dpi). The above results indicate that overexpression of BnaC.CCR2.b leads to an increase in lignin content in the stems, which subsequently leads to increased resistance to S. sclerotiorum. Our findings demonstrate that increasing the lignin content in the stems of B. napus is an important strategy for controlling Sclerotinia.

RESUMEN

BACKGROUND: Lignification of secondary cell walls is a major factor conferring recalcitrance of lignocellulosic biomass to deconstruction for fuels and chemicals. Genetic modification can reduce lignin content and enhance saccharification efficiency, but usually at the cost of moderate-to-severe growth penalties. We have developed a method, using a single DNA construct that uses CRISPR-Cas9 gene editing to knock-out expression of an endogenous gene of lignin monomer biosynthesis while at the same time expressing a modified version of the gene's open reading frame that escapes cutting by the Cas9 system and complements the introduced mutation in a tissue-specific manner. RESULTS: Expressing the complementing open reading frame in vessels allows for the regeneration of Arabidopsis plants with reduced lignin, wild-type biomass yield, and up to fourfold enhancement of cell wall sugar yield per plant. The above phenotypes are seen in both homozygous and bi-allelic heterozygous T1 lines, and are stable over at least four generations. CONCLUSIONS: The method provides a rapid approach for generating reduced lignin trees or crops with one single transformation event, and, paired with a range of tissue-specific promoters, provides a general strategy for optimizing loss-of-function traits that are associated with growth penalties. This method should be applicable to any plant species in which transformation and gene editing are feasible and validated vessel-specific promoters are available.

RESUMEN

Background: Sclerotinia sclerotiorum (Ss) is a broad host range necrotrophic ascomycete fungus affecting over 400 plant species. Ss causes stem rot disease on Camelina sativa (Cs) an allohexaploid crucifer species that is promoted as a low input crop and industrial oil attributes suitable as biofuel and lubricant. Histochemical and molecular studies has linked resistance to Ss in C. sativa with the cell wall lignification (Eynck et al., 2012) and reported constitutive expression of Cinnamoyl-CoA Reductase 4 (CsCCR4) gene, in the Cs resistant line CN114263. Modern breeding efforts, such as gene editing, are needed to improve commercial lines and to limit the risk of crop loss which would be substantial to producers. Objectives: To investigate the importance of monolignol biosynthesis and the role of CsCCR4 in Camelina resistance to Ss we generated CsCCR4 knockout mutants of CN114263 Camelina line using CRISPR/Cas9-mediated gene editing. Materials and Methods: Thirty T1 plants were produced via floral dip transformation followed by glyphosate spraying that was used in the first step of screening procedures and were confirmed by PCR method. Transgene's T-DNA copy number variation, T-DNA CNV, in T1 and T2 progenitors were determined using digital droplet PCR (ddPCR) and the occurrence of mutation in the three copies of CsCCR4 homeologues in T1 and T2 generations were scrutinized by drop-off assay technique. To make sure that if the created mutants in T2 plants are real, TOPO TA sequencing flanking the Cas9/gRNA specific hot point of cleavage for three of them was conducted. Results: In the T1 generation, 25 plants were confirmed which had between one to nine T-DNA copies in the corresponding Camelina genome. In T2 generation the population were screened for potential mutation in CsCCR4 gene. Various types of mutations, including insertions and deletions, were demonstrated in three copies of CsCCR4. In fact, CRISPR system could have cut one, two or three copies of the gene in events numbered T2-plant 10, T2-plant 15 and T2-plant 19, respectively. The T3-plant 19 which showed mutation in all versions of CsCCR4 in previous generation had susceptibility to S. sclerotiorum invasion and was kept as real csccr4 mutant material for further investigations of Camelina-Sclerotinia interaction. Mutation in CsCCR4 had occurred through error-prone none- homologous end joining (NHEJ) nucleus DNA repair pathway. Ss challenge on the early flowering T3 generation. The T3 plants with mutation causing premature stop codon at position 217 of CsCCR4 were compromised in their resistance to Ss compared to the wildtype resistant control parent CN114263. Conclusion: Using ddPCR it easily was possible to identify both the T-DNA CNV and occurrence of mutation in CsCCR4 homeologues in T1 and T2 progenitors. We illustrated that CRISPR/Cas9-mediated mutation is a decent technique that can be utilized to expedite the mutant line development which could assist to figure out the activity of a CsCCR4 gene in defense responses to the pathogens in C. sativa as prospective oilseed crop for biodiesel production.

RESUMEN

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

Asunto(s)

RESUMEN

BACKGROUND: The content of stone cells and lignin is one of the key factors affecting the quality of pear fruit. In a previous study, we determined the developmental regularity of stone cells and lignin in 'Dangshan Su' pear fruit 15-145 days after pollination (DAP). However, the development of fruit stone cells and lignin before 15 DAP has not been heavily researched. RESULTS: In this study, we found that primordial stone cells began to appear at 7 DAP and that the fruit had formed a large number of stone cells at 15 DAP. Subsequently, transcriptome sequencing was performed on fruits at 0, 7, and 15 DAP and identified 3834 (0 vs. 7 DAP), 4049 (7 vs. 15 DAP) and 5763 (0 vs. 15 DAP) DEGs. During the 7-15 DAP period, a large number of key enzyme genes essential for lignin biosynthesis are gradually up-regulated, and their expression pattern is consistent with the accumulation of lignin in this period. Further analysis found that the biosynthesis of S-type lignin in 'Dangshan Su' pear does not depend on the catalytic activity of PbSAD but is primarily generated by the catalytic activity of caffeoyl-CoA through CCoAOMT, CCR, F5H, and CAD. We cloned PbCCR1, 2 and analysed their functions in Chinese white pear lignin biosynthesis. PbCCR1 and 2 have a degree of functional redundancy; both demonstrate the ability to participate in lignin biosynthesis. However, PbCCR1 may be the major gene for lignin biosynthesis, while PbCCR2 has little effect on lignin biosynthesis. CONCLUSIONS: Our results revealed that 'Dangshan Su' pear began to form a large number of stone cells and produce lignin after 7 DAP and mainly accumulated materials from 0 to 7 DAP. PbCCR1 is mainly involved in the biosynthesis of lignin in 'Dangshan Su' pear and plays a positive role in lignin biosynthesis.

Asunto(s)

RESUMEN

Since lignin greatly affects stem strength, which is an important agronomical trait, understanding the relationship between lodging resistance and lignin synthesis is important in barley breeding and selection processes. The aim of the study was to reveal the connection between physiological aspects of lignin synthesis and genetic background of barley cultivars with different lodging phenotype. Three barley cultivars Astor, Scarlett and Jaran were compared by measuring lignin, cellulose and total soluble phenolics content, phenylalanine ammonia-lyase activity (PAL) and expression of cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) in three lower internodes at flowering and grain filling stage. To assess their genetic background simple sequence repeats (SSR) markers, connected to lodging resistance and plant height, were analyzed. Compared to lodging susceptible cultivars Scarlett and Jaran, a lodging resistant cultivar Astor revealed different dynamics of lignin synthesis and deposition, showing higher PAL activity and total soluble phenolics content as well as higher expression of CCR and CAD genes in the second internode at grain filling stage. Analysis of SSR markers associated with quantitative trait loci (QTL) for lodging resistance revealed that Astor discriminates from Scarlett and Jaran by marker Bmag337 connected with elongation of the second internode. Lignification process is under a strong influence of genotype and environmental factors which determine lignin synthesis dynamics and deposition of lignin in the cell walls of barley.

Asunto(s)

RESUMEN

Dihydroflavonol-4-reductase (DFR) is a key enzyme in the reduction of dihydroflavonols to leucoanthocyanidins in both anthocyanin biosynthesis and proanthocyanidin accumulation. In many plant species, it is encoded by a gene family, however, how the different copies evolve either to function in different tissues or at different times or to specialize in the use of different but related substrates needs to be further investigated, especially in monocot plants. In this study, a total of eight putative DFR-like genes were firstly cloned from Freesia hybrida. Phylogenetic analysis showed that they were classified into different branches, and FhDFR1, FhDFR2, and FhDFR3 were clustered into DFR subgroup, whereas others fell into the group with cinnamoyl-CoA reductase (CCR) proteins. Then, the functions of the three FhDFR genes were further characterized. Different spatio-temporal transcription patterns and levels were observed, indicating that the duplicated FhDFR genes might function divergently. After introducing them into Arabidopsis dfr (tt3-1) mutant plants, partial complementation of the loss of cyanidin derivative synthesis was observed, implying that FhDFRs could convert dihydroquercetin to leucocyanidin in planta. Biochemical assays also showed that FhDFR1, FhDFR2, and FhDFR3 could utilize dihydromyricetin to generate leucodelphinidin, while FhDFR2 could also catalyze the formation of leucocyanidin from dihydrocyanidin. On the contrary, neither transgenic nor biochemical analysis demonstrated that FhDFR proteins could reduce dihydrokaempferol to leucopelargonidin. These results were consistent with the freesia flower anthocyanin profiles, among which delphinidin derivatives were predominant, with minor quantities of cyanidin derivatives and undetectable pelargonidin derivatives. Thus, it can be deduced that substrate specificities of DFRs were the determinant for the categories of anthocyanins aglycons accumulated in F. hybrida. Furthermore, we also found that the divergence of the expression patterns for FhDFR genes might be controlled at transcriptional level, as the expression of FhDFR1/FhDFR2 and FhDFR3 was controlled by a potential MBW regulatory complex with different activation efficiencies. Therefore, it can be concluded that the DFR-like genes from F. hybrida have diverged during evolution to play partially overlapping roles in the flavonoid biosynthesis, and the results will contribute to the study of evolution of DFR gene families in angiosperms, especially for monocot plants.

RESUMEN

Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as CCR and CCR-like genes, collectively called OsCCRs. To elucidate the functions of OsCCRs, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 OsCCRs, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in Escherichia coli and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that OsCCR20 was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that OsCCR20 is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of OsCCR17 and 21 were induced in response to biotic and abiotic stresses, such as Magnaporthe grisea and Xanthomonas oryzae pv. oryzae (Xoo) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.

RESUMEN

After initiation, leaves first undergo rapid cell proliferation. During subsequent development, leaf cells gradually exit the proliferation phase and enter the expansion stage, following a basipetally ordered pattern starting at the leaf tip. The molecular mechanism directing this pattern of leaf development is as yet poorly understood. By genetic screening and characterization of Arabidopsis mutants defective in exit from cell proliferation, we show that the product of the CINNAMOYL CoA REDUCTASE (CCR1) gene, which is required for lignin biosynthesis, participates in the process of cell proliferation exit in leaves. CCR1 is expressed basipetally in the leaf, and ccr1 mutants exhibited multiple abnormalities, including increased cell proliferation. The ccr1 phenotypes are not due to the reduced lignin content, but instead are due to the dramatically increased level of ferulic acid (FeA), an intermediate in lignin biosynthesis. FeA is known to have antioxidant activity, and the levels of reactive oxygen species (ROS) in ccr1 were markedly reduced. We also characterized another double mutant in CAFFEIC ACID O-METHYLTRANSFERASE (comt) and CAFFEOYL CoA 3-O-METHYLTRANSFERASE (ccoaomt), in which the FeA level was dramatically reduced. Cell proliferation in comt ccoaomt leaves was decreased, accompanied by elevated ROS levels, and the mutant phenotypes were partially rescued by treatment with FeA or another antioxidant (N-acetyl-L-cysteine). Taken together, our results suggest that CCR1, FeA and ROS coordinate cell proliferation exit in normal leaf development.

Asunto(s)

RESUMEN

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

RESUMEN

In monocots, lignin content has a strong impact on the digestibility of the cell wall fraction. Engineering lignin biosynthesis requires a profound knowledge of the role of paralogues in the multigene families that constitute the monolignol biosynthesis pathway. We applied a bioinformatics approach for genome-wide identification of candidate genes in Lolium perenne that are likely to be involved in the biosynthesis of monolignols. More specifically, we performed functional subtyping of phylogenetic clades in four multigene families: 4CL, COMT, CAD and CCR. Essential residues were considered for functional clade delineation within these families. This classification was complemented with previously published experimental evidence on gene expression, gene function and enzymatic activity in closely related crops and model species. This allowed us to assign functions to novel identified L. perenne genes, and to assess functional redundancy among paralogues. We found that two 4CL paralogues, two COMT paralogues, three CCR paralogues and one CAD gene are prime targets for genetic studies to engineer developmentally regulated lignin in this species. Based on the delineation of sequence conservation between paralogues and a first analysis of allelic diversity, we discuss possibilities to further study the roles of these paralogues in lignin biosynthesis, including expression analysis, reverse genetics and forward genetics, such as association mapping. We propose criteria to prioritise paralogues within multigene families and certain SNPs within these genes for developing genotyping assays or increasing power in association mapping studies. Although L. perenne was the target of the analyses presented here, this functional subtyping of phylogenetic clades represents a valuable tool for studies investigating monolignol biosynthesis genes in other monocot species.

Asunto(s)

RESUMEN

Volatile phenylpropenes play important roles in the mediation of interactions between plants and their biotic environments. Their biosynthesis involves the elimination of the oxygen functionality at the side-chain of monolignols and competes with lignin formation for monolignol utilization. We hypothesized that biochemical steps before the monolignol branch point are shared between phenylpropene and lignin biosynthesis; however, genetic evidence for this shared pathway has been missing until now. Our hypothesis was tested by RNAi suppression of the petunia (Petunia hybrida) cinnamoyl-CoA reductase 1 (PhCCR1), which catalyzes the first committed step in monolignol biosynthesis. Detailed metabolic profiling and isotopic labeling experiments were performed in petunia transgenic lines. Downregulation of PhCCR1 resulted in reduced amounts of total lignin and decreased flux towards phenylpropenes, whereas internal and emitted pools of phenylpropenes remained unaffected. Surprisingly, PhCCR1 silencing increased fluxes through the general phenylpropanoid pathway by upregulating the expression of cinnamate-4-hydroxylase (C4H), which catalyzes the second reaction in the phenylpropanoid pathway. In conclusion, our results show that PhCCR1 is involved in both the biosynthesis of phenylpropenes and lignin production. However, PhCCR1 does not perform a rate-limiting step in the biosynthesis of phenylpropenes, suggesting that scent biosynthesis is prioritized over lignin formation in petals.

Asunto(s)