Lignin Accumulation Research Articles

Formulation of the encapsulated rhizospheric Ochrobactrum ciceri supplemented with alginate for potential antifungal activity against the chili collar rot pathogen

The collar rot disease caused by the soil-borne fungus Sclerotium rolfsii (Sacc.) is a major constraint of chili (Capsicum annum L.) production in Pakistan. Considering the fact that biological control has low efficacy in field conditions, their encapsulation into biopolymers like alginate meets the essential criteria for bacteria viability, effectiveness, shelf life, stability, and controlled release. The current research was conducted to develop stable alginate beads of biocontrol bacteria (Ochrobactrum ciceri) and to check their efficacy in managing collar rot disease in C. annum. In vitro, antifungal bioassays indicated that increasing concentrations (16, 24, and 64%) of culture filtrate or the cell pellets significantly decreased fungal growth and enzyme activity (catalase: CAT, peroxidase: POX, polyphenol oxidase: PPO, and phenylalanine ammonia-lyase: PAL), hence increased offshoots hyphae, and caused distortion in sclerotia and hyphae. O. ciceri successfully developed stable alginate beads (AlgB-OC) as indicated by FTIR analysis, encapsulation efficiency (91.15%), moisture content (99.19%), swelling ratio (130%), particle size (wet: 2.11 mm; dry: 1.15 mm), film-forming time (48 h), and slow-release of entrapped bacteria till 30 days. AlgB-OC exhibited 76% antifungal potential in vitro and managed 70% of the collar rot disease in vivo. Moreover, the application of AlgB-OC significantly improved plant growth (length and biomass) and physio-chemical attributes (photosynthetic pigments, total protein content, activity of CAT, POX, PPO, and PAL), along with more lignin, phenolics, gel, and starch accumulation in roots. Multivariate analysis based on a total of 13 morpho-physiological indices of chili plants also identified AlgB-OC as the most effective treatment for disease management. It was concluded that the formulation of O. ciceri into alginate could be an effective alternative for managing collar rot disease in chili plants and obtaining better plant growth.

Molecular hydrogen-based irrigation extends strawberry shelf life by improving the synthesis of cell wall components in fruit

Strawberries (Fragaria × ananassa Duch.) are highly perishable due to their soft texture, and easily decay during transportation and storage. Our previous report showed that molecular hydrogen-based irrigation in the form of hydrogen nanobubble water (HNW) could obviously improve flavor and consumer preferences of strawberry fruit at the harvesting stage. This study further investigated whether this approach could influence the shelf life of strawberry. During storage for 15 d at 4 ℃, HNW delayed the degradation in the quality of strawberry fruit, evaluated by alteration in sensory attributes (including aroma, luster, color, shape, and general evaluation), weight loss, contents of vitamin C, soluble sugar, and titratable acidity, and sugar/acid ratio. The higher levels of lignin, cellulose, and hemicellulose were observed. Compared to conventional surface water (control) irrigation, the firmness of fruit was increased by HNW irrigation at the harvest stage, and progressively maintained during the storage period. Molecular evidence revealed that before the harvesting period, the transcripts of cinnamyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), as well as cellulose synthase 1/4/6 (CesF1/4/6), the representative genes for the synthesis of lignin, cellulose, and hemicellulose, were induced by HNW irrigation. These changes were positively matched with the increased lignin, cellulose, and hemicellulose contents at the harvesting period. Overall, the extended shelf life of strawberries achieved by molecular hydrogen-based irrigation might be due to the improvement in lignin, cellulose, and hemicellulose accumulation. Therefore, this study demonstrated the effectiveness of HNW irrigation for retarding the decay of harvested strawberries.

Source to Sink of Lignin Phenols in a Subtropical Forest of Southwest China

In biodiverse forest ecosystems, plant diversity has been reported to increase plant-derived lignin accumulation and soil organic carbon (SOC) storage. However, little is known about the fate of lignin and its degradation dynamics from plant to soil. This process is critical for the formation of SOC, especially in natural forest ecosystems with diverse plant species. This study presents the lignin biomarker characteristics of several common plant species and in mixed litter. The study was conducted in 45 plots along a plant species diversity gradient in a subtropical forest located in southwest China. Our results demonstrate that lignin content and its biochemical characteristics in plant leaves vary among species, while different plant species also alter the content of lignin and its monomeric compounds in the litter. Lignin compounds are gradually disintegrated from plant leaf to litter and then to soil, further indicating that plant-derived lignin from plant sources contributes to the formation and accumulation of forest SOC. These findings provide novel information on the linkage between tree species diversity and lignin accumulation while indicating the role of plant-derived lignin on SOC storage. These results may be useful in predicting forest soil C dynamics in Earth system models.

Putrescine (1,4-Diaminobutane) enhances antifungal activity in postharvest mango fruit against Colletotrichum gloeosporioides through direct fungicidal and induced resistance mechanisms

Anthracnose decay caused by Colletotrichum gloeosporioides greatly shortens the shelf life and commercial quality of mango fruit. Putrescine (1,4-Diaminobutane) is involved in modulating plant defense to various environmental stresses. In this research, in vivo and in vitro tests were used to explore the antifungal activity and the underlying mechanism of putrescine against C. gloeosporioides in mango fruit after harvested. In vivo tests suggested that putrescine markedly delayed the occurrence of disease and limited the spots expansion on inoculated mango fruit. Further analysis exhibited that putrescine treatment enhanced disease resistance, along with enhanced activities of chitinase (CHI), β-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), polyphenol oxidase (PPO) and the accumulation of lignin, flavonoid, phenolics, and anthocyanin in infected mango fruit. In addition, in vitro tests showed that putrescine exerted strongly antifungal activity against C. gloeosporioides. Putrescine induced the production of reactive oxygen species (ROS) and severe lipid peroxidation damage in C. gloeosporioides mycelia, resulting in the leakage of soluble protein, soluble sugar, nucleic acids, K+ and Ca2+ of C. gloeosporioides mycelia. The mycelium treated with putrescine showed severe deformity and shrinkage, and even cracking. Taken together, putrescine could effectively reduce the incidence rate and severity of anthracnose disease possibly through direct fungicidal effect and indirect induced resistance mechanism, thus showing great potential to be applied to disease control.

Overexpression of a laccase gene, DiLAC17, from Davidia involucrata causes severe seed abortion in Arabidopsis

Seed abortion is a common phenomenon in woody plants, especially in rare and endangered species. Serious seed abortion occurs in the dove tree and largely restricts its natural reproduction. A number of differentially expressed genes (DEGs) between normal and aborted seeds of the dove tree have been previously identified through transcriptome profiling. Among these, most DEGs encoding laccase showed significant upregulation in the aborted seeds. In this study, the laccase gene with the highest expression level in aborted seeds, DiLAC17, was cloned from the dove tree genome and further verified. Overexpression of the DiLAC17 gene in Arabidopsis resulted in retarded growth, deformed siliques, and severe seed abortion. Most Arabidopsis genes involved in seed development, such as AtLEC2, AtANT1, and AtRGE1, were suppressed in the transgenic lines. Laccase activity and lignin content were significantly improved in transgenic lines under ectopic overexpression of the DiLAC17 gene. Excessive lignin accumulation in the early developmental stage was assumed to be a key cause of restricting silique growth and seed expansion, which ultimately led to seed abortion. These results indicate a laccase-mediated pathway for seed abortion, which might be a strategy adopted by this rare and endangered species to reduce the reproductive load.

PoWRKY17 promotes drought tolerance in Paeonia ostii by modulating lignin accumulation

Drought is a detrimental environmental factor limiting sustainable agricultural development worldwide. Paeonia ostii, an oil-producing woody crop, while its endogenous mystery underlying this tolerance remains largely elusive to scholars. Herein, we reported that PoWRKY17, a WRKY transcription factor from IId subgroup, positively functioned in face of drought stress in P. ostii. PoWRKY17 expressed in significantly higher abundance when drought was applied. Silencing of PoWRKY17 substantially reduced drought resistance, with decreased leaf water content and protective enzyme activities, increased relative electric conductivity (REC), malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation. On the contrary, overexpression of PoWRKY17 endowed plants with higher drought tolerance with opposite drought-relevant physiological indices. Notably, both PoWRKY17-silenced P. ostii and PoWRKY17-overexpressing plants showed changes in lignin content under drought stress. Based on this clue, we quantified the expression of lignin biosynthetic caffeoyl-CoA O-methyltransferase (CCoAOMT) gene in PoWRKY17-silenced P. ostii and found that PoCCoAOMT expression was significantly repressed. Next, protein-gene interaction assays showed that PoWRKY17 interacted with PoCCoAOMT promoter by targeting the W-box element and activated PoCCoAOMT expression under drought stress, which has been early verified to act as a positive participant in P. ostii drought tolerance. This study broadens our understanding of WRKY-mediated lignin biosynthesis regulation in response to drought stress in plants.

Alterations in plant anatomy and higher lignin synthesis provides drought tolerance in cluster bean [Cyamopsis tetragonoloba (L.) Taub.

Four contrasting varieties of guar, RGC-1002 and RGC-1038, drought tolerant, while, Sarada and RGC-936, drought sensitive, were monitored in watered and drought. The water status, phenolics, plant anatomy and transcript level of genes related to anatomical traits were assessed. The study aimed to decipher the anatomical adaptations of guar plants in response to water stress. The physiological determinants, relative water content (RWC), water potential (ψ), and leaf membrane damage, declined under drought in all four varieties although, the decrement was lesser in the tolerant varieties. Furthermore, the tolerant cultivars subjected to water stress recorded higher accumulation of total phenolic content, anthocyanin and lignin, which efficiently scavenge the reactive oxygen species. The results suggest that the cultivars RGC-1002 and RGC-1038 are better able to resist drought-induced oxidative stress than Sarada and RGC-936. Moreover, leaf, petiole, stem and root anatomical traits viz. size of epidermal cell, parenchyma, width of cortex layer, and diameter of xylem vessels were narrowed in all the varieties although, the decrement was lesser in the tolerant varieties under drought. The expression analysis of genes revealed that drought-tolerant varieties showed enhanced mechanical support for water conduction by up-regulation of genes, Phenylalanine ammonia-lyase1 (PAL1), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), cinnamoyl-CoA reductase (CCR), caffeoyl-CoA O-methyltransferase (CCOMT), and cinnamyl-alcohol dehydrogenase (CAD6) in water stress conditions. The alterations in physio-anatomical, biochemical and gene expression traits in tolerant guar varieties enabled them to maintain steady nutrient transport while reducing the risk of embolisms and increasing water-flow resistance for better survival in water stressed conditions.

Methyl jasmonate‐dependent hydrogen sulfide mediates melatonin‐induced aphid resistance of watermelon

AbstractAs one of the most destructive pests, aphids cause significant damage on various agricultural and horticultural crops. Recently, melatonin has been shown to enhance plant resistance to aphids; however, the underlying mechanisms remain unclear. In this study, our results showed that melatonin, MeJA, and H2S enhanced aphid resistance of watermelon in a dose‐dependent manner, accompanied by increases in the defense‐related enzyme activities and lignin accumulation. On the plants pretreated with 100 μM melatonin, 100 μM MeJA, and 50 μM NaHS, the numbers of aphids were 86.0%, 59.6%, and 47.9% lower than that on control plants, respectively, after aphid infestation for 7 days. Melatonin application induced MeJA and H2S accumulation in response to aphid infestation, while inhibition of MeJA and H2S accumulation attenuated or abolished melatonin‐induced defense response and aphid resistance, suggestive of the involvement of MeJA and H2S in melatonin‐induced aphid resistance. MeJA also increased H2S accumulation, but inhibition of MeJA biosynthesis prevented melatonin‐enhanced H2S accumulation, suggesting that MeJA mediates melatonin‐induced H2S accumulation. Furthermore, inhibition of H2S production attenuated MeJA‐induced defense response and aphid resistance. Taken together, the current study reveals a novel mechanism in which MeJA‐dependent H2S signaling is involved in melatonin‐induced defense response and subsequent aphid resistance. The increasing concern to minimize the use of pesticides and to switch onto sustainable and natural control strategies indicates the great exploitation of such a mechanism in combating aphids.

Integrated transcriptomic and metabolomic analysis reveals the effects of polyploidization on the lignin content and metabolic pathway in Eucalyptus

BackgroundLignin is a major restriction factor for the industrial production of biomass resources, such as pulp and bioenergy. Eucalyptus is one of the most important sources of pulp and bioenergy. After polyploidization, the lignin content of forest trees is generally reduced, which is considered a beneficial genetic improvement. However, the differences in the lignin content between triploid and diploid Eucalyptus and the underlying regulatory mechanism are still unclear.ResultsWe conducted a comprehensive analysis at the phenotypic, transcriptional and metabolite levels between Eucalyptus urophylla triploids and diploids to reveal the effects of polyploidization on the lignin content and lignin metabolic pathway. The results showed that the lignin content of Eucalyptus urophylla triploid stems was significantly lower than that of diploids. Lignin-related metabolites were differentially accumulated between triploids and diploids, among which coniferaldehyde, p-coumaryl alcohol, sinapaldehyde and coniferyl alcohol had significant positive correlations with lignin content, indicating that they might be primarily contributing metabolites. Most lignin biosynthetic genes were significantly downregulated, among which 11 genes were significantly positively correlated with the lignin content and above metabolites. Furthermore, we constructed a co-expression network between lignin biosynthetic genes and transcription factors based on weighted gene co-expression network analysis. The network identified some putative orthologues of secondary cell wall (SCW)-related transcription factors, among which MYB52, MYB42, NAC076, and LBD15 were significantly downregulated in Eucalyptus urophylla triploids. In addition, potential important transcription factors, including HSL1, BEE3, HHO3, and NAC046, also had high degrees of connectivity and high edge weights with lignin biosynthetic genes, indicating that they might also be involved in the variation of lignin accumulation between triploid and diploid Eucalyptus urophylla.ConclusionsThe results demonstrated that some lignin-related metabolites, lignin biosynthetic genes and transcription factors in Eucalyptus urophylla triploids may be relatively sensitive in response to the polyploidization effect, significantly changing their expression levels, which ultimately correlated with the varied lignin content. The analysis of the underlying formation mechanism could provide beneficial information for the development and utilization of polyploid biomass resources, which will be also valuable for genetic improvement in other bioenergy plants.

Genome-Wide Identification of WRKY Gene Family and Functional Characterization of CcWRKY25 in Capsicum chinense.

Pepper is renowned worldwide for its distinctive spicy flavor. While the gene expression characteristics of the capsaicinoid biosynthesis pathway have been extensively studied, there are already a few reports regarding transcriptional regulation in capsaicin biosynthesis. In this study, 73 WRKYs were identified in the genome of Capsicum chinense, and their physicochemical traits, DNA, and protein sequence characteristics were found to be complex. Combining RNA-seq and qRT-PCR data, the WRKY transcription factor CA06g13580, which was associated with the accumulation tendency of capsaicinoid, was screened and named CcWRKY25. CcWRKY25 was highly expressed in the placenta of spicy peppers. The heterologous expression of CcWRKY25 in Arabidopsis promoted the expression of genes PAL, 4CL1, 4CL2, 4CL3, CCR, and CCoAOMT and led to the accumulation of lignin and flavonoids. Furthermore, the expression of the capsaicinoid biosynthesis pathway genes (CBGs) pAMT, AT3, and KAS was significantly reduced in CcWRKY25-silenced pepper plants, resulting in a decrease in the amount of capsaicin. However, there was no noticeable difference in lignin accumulation. The findings suggested that CcWRKY25 could be involved in regulating capsaicinoid synthesis by promoting the expression of genes upstream of the phenylpropanoid pathway and inhibiting CBGs' expression. Moreover, the results highlighted the role of CcWRKY25 in controlling the pungency of pepper and suggested that the competitive relationship between lignin and capsaicin could also regulate the spiciness of the pepper.

Uncovering mechanisms governing stem growth in peanut (Arachis hypogaea L.) with varying plant heights through integrated transcriptome and metabolomics analyses

The mechanisms responsible for stem growth in peanut (Arachis hypogaea L.) cultivars with varying plant heights remain unclear, despite the significant impact of plant height on peanut yield. Therefore, this study aimed to investigate the underlying mechanisms of peanut stem growth using phenotypic, physiological, transcriptomic, and metabolomic analyses. The findings revealed that the tallest cultivar, HY33, exhibited the highest rate of stem growth and accumulated the most stem dry matter, followed by the intermediate cultivar, SH108, while the dwarf cultivar, Df216, displayed the lowest values. Furthermore, SH108 exhibited a higher harvest index, as well as superior pod and kernel yields compared to both HY33 and Df216. Transcriptome and metabolome analyses identified differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs) associated with phenylpropanoid and flavonoid biosynthesis. Notably, downregulated DEGs in Df216/HY33 and Df216/SH108 included phenylalanine ammonia-lyase (PAL), caffeoyl-CoA O-methyltransferase (COMT), and ferulate-5-hydroxylase (F5H), while downregulated DEMs included p-coumaryl alcohol, chlorogenic acid, and L-epicatechin. Compared to HY33, the reduced activities of PAL, COMT, and F5H resulted in a decreased stem lignin content in Df216. Additionally, downregulated DEGs involved in gibberellin (GA) and brassinosteroid (BR) biosynthesis were identified in Df216/HY33, which contributed to the lowest levels of GA1, GA3, and BR contents in Df216. The results suggest that the dwarf phenotype arises from impaired GA and BR biosynthesis and signaling, resulting in a slower stem growth rate and reduced lignin accumulation.

Heterogeneous population distribution enhances resistance to wheat lodging by optimizing the light environment

Lodging is still the key factor that limits continuous increases in wheat yields today, because the mechanical strength of culms is reduced due to low-light stress in populations under high-yield cultivation. The mechanical properties of the culm are mainly determined by lignin, which is affected by the light environment. However, little is known about whether the light environment can be sufficiently improved by changing the population distribution to inhibit culm lodging. Therefore, in this study, we used the wheat cultivar “Xinong 979” to establish a low-density homogeneous distribution treatment (LD), high-density homogeneous distribution treatment (HD), and high-density heterogeneous distribution treatment (HD-h) to study the regulatory effects and mechanism responsible for differences in the lodging resistance of wheat culms under different population distributions. Compared with LD, HD significantly reduced the light transmittance in the middle and basal layers of the canopy, the net photosynthetic rate in the middle and lower leaves of plants, the accumulation of lignin in the culm, and the breaking resistance of the culm, and thus the lodging index values increased significantly, with lodging rates of 67.5% in 2020–2021 and 59.3% in 2021–2022. Under HD-h, the light transmittance and other indicators in the middle and basal canopy layers were significantly higher than those under HD, and the lodging index decreased to the point that no lodging occurred. Compared with LD, the activities of phenylalanine ammonia-Lyase (PAL), 4-coumarate: coenzyme A ligase (4CL), catechol-O-methyltransferase (COMT), and cinnamyl-alcohol dehydrogenase (CAD) in the lignin synthesis pathway were significantly reduced in the culms under HD during the critical period for culm formation, and the relative expression levels of TaPAL, Ta4CL, TaCOMT, and TaCAD were significantly downregulated. However, the activities of lignin synthesis-related enzymes and their gene expression levels were significantly increased under HD-h compared with HD. A partial least squares path modeling analysis found significant positive effects between the canopy light environment, the photosynthetic capacity of the middle and lower leaves of plants, lignin synthesis and accumulation, and lodging resistance in the culms. Thus, under conventional high-density planting, the risk of wheat lodging was significantly higher. Accordingly, the canopy light environment can be optimized by changing the heterogeneity of the population distribution to improve the photosynthetic capacity of the middle and lower leaves of plants, promote lignin accumulation in the culm, and enhance lodging resistance in wheat. These findings provide a basis for understanding the mechanism responsible for the lower mechanical strength of the culm under high-yield wheat cultivation, and a theoretical basis and for developing technical measures to enhance lodging resistance.

Two R2R3-MYB transcription factors from Chinese cedar (Cryptomeria fortunei Hooibrenk) are involved in the regulation of secondary cell wall formation

As the most abundant renewable energy source, wood comprises the secondary cell wall (SCW). SCW biosynthesis involves lignin and cellulose deposition. Increasing studies have illustrated that R2R3-MYB transcription factors (TFs) play pivotal roles in affecting lignin accumulation and SCW formation. Nevertheless, the regulatory roles of R2R3-MYBs are still unresolved in Cryptomeria fortunei Hooibrenk cambium and wood formation. To dissect the potentials of CfMYBs, we successfully cloned and intensively studied the functions of CfMYB4 and CfMYB5 in SCW formation and abiotic stress response. They both contained the conserved MYB domain capable of forming a special structure that could bind to the core motifs of downstream genes. The phylogenetic tree implied that two CfMYBs clustered into different evolutionary branches. They were predominantly expressed in the stem and were localized to the nucleus. Furthermore, CfMYB4 functioned as an activator to enhance lignin and cellulose accumulation, and increase the SCW thickness by elevating the expression levels of SCW-related genes. By contrast, CfMYB5 negatively regulated lignin and cellulose biosynthesis, and decreased SCW formation by reducing the expression of SCW biosynthetic genes. Our data not only highlight the regulatory functions of CfMYBs in lignin deposition but also provide critical insights into the development of strategies for the genetic improvement of Cryptomeria fortunei wood biomass.

CtCYP71A1 promotes drought stress tolerance and lignin accumulation in safflower and Arabidopsis

Lignin is a complex phenolic polymer that forms interwoven networks in the cell wall and is crucial to plant development and stress responses. The root tissues of safflower contain a high concentration of lignin, which may contribute to drought regulation mechanism in safflower. Cytochrome P450 is the largest oxidase family, which is often dubbed with a significant role in plant metabolism. However, little is known about the molecular mechanism of CYP450-mediated regulation of drought stress tolerance in safflower roots. In this study, a putative CtCYP71A1 gene from safflower was identified by combining genome-wide and transcriptomic analysis. In safflower, a total of 16 CtCYP71A enzyme coding genes were identified. The conserved structural topology of CtCYP71As reveals several signature motifs that are crucial to plant growth and stress response. The expression of CtCYP71A1 was shown to be diversely expressed in different tissues, however, it was significantly up-regulated in roots under normal condition. Similarly, in response to PEG stress, the expression of CtCYP71A1 in roots gradually increased, whereas it was upregulated initially and subsequently downregulated with ABA, GA3, and SA stress. In addition, the phenotypic analysis and root morphology of CtCYP71A1 overexpressed Arabidopsis lines showed enhanced drought tolerance with improved lignin content than that of wild-type and antisense plants, which was further confirmed by expression analysis, and various physiological indexes. Similarly, overexpression of CtCYP71A1 resulted in the positive regulation of several other genes involved in lignin biosynthesis compared to WT and antisense plants. Furthermore, drought sensitivity was observed in safflower roots with CtCYP71A1 virus-induced gene silencing assay. Together, these findings revealed important insights into the crucial role of CtCYP71A1 in drought tolerance via lignin accumulation in safflower.

Genome-wide identification and expression analysis of MYB gene family in Cajanus cajan and CcMYB107 improves plant drought tolerance.

MYB transcription factor (TF) is one of the largest superfamilies that play a vital role in multiple plant biological processes. However, the MYB family has not been comprehensively identified and functionally verified in Cajanus cajan, which is the sixth important legume crop. Here, 170 CcR2R3-MYBs were identified and divided into 43 functional subgroups. Segmental and tandem duplications and alternative splicing events were found and promoted the expansion of the CcR2R3-MYB gene family. Functional prediction results showed that CcR2R3-MYBs were mainly involved in secondary metabolism, cell fate and identity, developmental processes, and responses to abiotic stress. Cis-acting element analysis of promoters revealed that stress response elements were widespread in the above four functional branches, further suggesting CcR2R3-MYBs were extensively involved in abiotic stress response. The transcriptome data and qRT-PCR results indicated that most of the CcR2R3-MYB genes responded to various stresses, of which the expression of CcMYB107 was significantly induced by drought stress. Overexpression of CcMYB107 enhanced antioxidant enzyme activity and increased proline and lignin accumulation, thus improving the drought resistance of Cajanus cajan. Furthermore, Overexpression of CcMYB107 up-regulated the expression of stress-related genes and lignin biosynthesis genes after drought stress. Our findings established a strong foundation for biological function investigation of CcR2R3-MYB TFs in Cajanus cajan. This article is protected by copyright. All rights reserved.

CsMYB15 positively regulates Cs4CL2-mediated lignin biosynthesis during juice sac granulation in navel orange.

'Lane Late', a late-maturing navel orange cultivar, is mainly distributed in the Three Gorges Reservoir area, which matures in the late March of the next year and needs overwintering cultivation. Citrus fruit granulation is a physiological disorder, which is characterized by lignification and dehydration of juice sac cells, seriously affecting the commercial value of citrus fruits. The pre-harvest granulation of late-maturing navel orange is main caused by low temperature in the winter, but its mechanism and regulation pattern remain unclear. In this study, a SG2-type R2R3-MYB transcription factor, CsMYB15, was identified from Citrus sinensis, which was significantly induced by both juice sac granulation and low temperature treatment. Subcellular localization analysis and transcriptional activation assay revealed that CsMYB15 protein was localized to the nucleus, and it exhibited transcriptional activation activity in yeast. Over-expression of CsMYB15 by stable transformation in navel orange calli and transient transformation in kumquat fruits and navel orange juice sacs significantly increased lignin content in the transgenic lines. Further, Yeast one hybrid, EMSA, and LUC assays demonstrated that CsMYB15 directly bound to the Cs4CL2 promoter and activated its expression, thereby causing a high accumulation of lignin in citrus. Taken together, these results elucidated the biological function of CsMYB15 in regulating Cs4CL2-mediated lignin biosynthesis, and provided novel insight into the transcriptional regulation mechanism underlying the juice sac granulation of late-maturing navel orange.

Transcriptomic and metabolomic analysis provides insights into lignin biosynthesis and accumulation and differences in lodging resistance in hybrid wheat

The use of hybrid wheat is one way to improve the yield in the future. However, greater plant heights increase lodging risk to some extent. In this study, two hybrid combinations with differences in lodging resistance were used to analyze the stem-related traits during the filling stage, and to investigate the mechanism of the difference in lodging resistance by analyzing lignin synthesis of the basal second internode (BSI). The stem-related traits such as the breaking strength, stem pole substantial degree (SPSD), and rind penetration strength (RPS), as well as the lignin content of the lodging-resistant combination (LRC), were significantly higher than those of the lodging-sensitive combination (LSC). The phenylpropanoid biosynthesis pathway was significantly and simultaneously enriched according to the transcriptomics and metabolomics analysis at the later filling stage. A total of 35 critical regulatory genes involved in the phenylpropanoid pathway were identified. Moreover, 42% of the identified genes were significantly and differentially expressed at the later grain-filling stage between the two combinations, among which more than 80% were strongly up-regulated at that stage in the LRC compared with LSC. On the contrary, the LRC displayed lower contents of lignin intermediate metabolites than the LSC. These results suggested that the key to the lodging resistance formation of LRC is largely the higher lignin synthesis at the later grain-filling stage. Finally, breeding strategies for synergistically improving plant height and lodging resistance of hybrid wheat were put forward by comparing the LRC with the conventional wheat applied in large areas.

Establishment of an efficient root mediated genetic transformation method for gene function verification in citrus

Although several methods of citrus genetic transformation have been studied and published, it is still necessary to further improve the efficiency of genetic transformation and increase the varieties used for citrus genetic transformation. Here, we report a simple, rapid and efficient root transformation technique for citrus to facilitate gene function studies. We found that infestation of Agrobacterium rhizogenes into citrus seedlings of five different species could produce stable transgenic roots in a short period after comparing and optimizing the selection of citrus species, bacterium concentration, cutting time and infection method. PCR and fluorescence observations showed a positive root transformation efficiency of approximately 13%-36% in the five tested citrus species. To demonstrate the utility of this method in the functional verification of citrus genes, a bimolecular fluorescence complementation (BiFC) assay was used to prove the interaction between CrCIPK6/9 and CrCBL5/6 in Citrus reticulata. Additionally, the PCR results showed that CrFT-RFP was transferred from the transgenic positive root to the stem in Citrus reticulata. Finally, overexpression of CjCAD from Citrus junos promoted lignin accumulation in transgenic citrus roots. This method could not only be used to study genes in roots but also various genes involved in plant growth and development. Taken together, we demonstrated that this method allows fast study of gene function in a wide range of citrus species, which would facilitate the understanding of novel genes in citrus.

Cell wall-related genes and lignin accumulation contribute to the root resistance in different maize (Zea mays L.) genotypes to Fusarium verticillioides (Sacc.) Nirenberg infection.

The fungal pathogen Fusarium verticillioides (Sacc.) Nirenberg (Fv) causes considerable agricultural and economic losses and is harmful to animal and human health. Fv can infect maize throughout its long agricultural cycle, and root infection drastically affects maize growth and yield. The root cell wall is the first physical and defensive barrier against soilborne pathogens such as Fv. This study compares two contrasting genotypes of maize (Zea mays L.) roots that are resistant (RES) or susceptible (SUS) to Fv infection by using transcriptomics, fluorescence, scanning electron microscopy analyses, and ddPCR. Seeds were infected with a highly virulent local Fv isolate. Although Fv infected both the RES and SUS genotypes, infection occurred faster in SUS, notably showing a difference of three to four days. In addition, root infections in RES were less severe in comparison to SUS infections. Comparative transcriptomics (rate +Fv/control) were performed seven days after inoculation (DAI). The analysis of differentially expressed genes (DEGs) in each rate revealed 733 and 559 unique transcripts that were significantly (P ≤0.05) up and downregulated in RES (+Fv/C) and SUS (+Fv/C), respectively. KEGG pathway enrichment analysis identified coumarin and furanocoumarin biosynthesis, phenylpropanoid biosynthesis, and plant-pathogen interaction pathways as being highly enriched with specific genes involved in cell wall modifications in the RES genotype, whereas the SUS genotype mainly displayed a repressed plant-pathogen interaction pathway and did not show any enriched cell wall genes. In particular, cell wall-related gene expression showed a higher level in RES than in SUS under Fv infection. Analysis of DEG abundance made it possible to identify transcripts involved in response to abiotic and biotic stresses, biosynthetic and catabolic processes, pectin biosynthesis, phenylpropanoid metabolism, and cell wall biosynthesis and organization. Root histological analysis in RES showed an increase in lignified cells in the sclerenchymatous hypodermis zone during Fv infection. These differences in the cell wall and lignification could be related to an enhanced degradation of the root hairs and the epidermis cell wall in SUS, as was visualized by SEM. These findings reveal that components of the root cell wall are important against Fv infection and possibly other soilborne phytopathogens.

Osmotic stress-induced lignin synthesis is regulated at multiple levels in alfalfa (Medicago sativa L.)

Alfalfa is an important forage crop. Yield and quality are frequently threatened by extreme environments such as drought and salt stress. As a component of the cell wall, lignin plays an important role in the abiotic stress response, the mechanisms of which have not been well clarified. In this study, we combined physiological, transcriptional, and metabolic analyses to reveal the changes in lignin content in alfalfa under mannitol-induced osmotic stress. Osmotic stress enhanced lignin accumulation by increasing G and S units, which was associated with increases in enzyme activities and decreases in 8 intermediate metabolites. Upon combined analysis of the transcriptome and metabolome, we identified five key structural genes and several coexpressed transcription factors, such as MYB and WRKY, which may play a core role in regulating lignin content and composition under osmotic stress. In addition, lignin synthesis was positively regulated by ABA but negatively regulated by ethylene under osmotic stress. These results provide new insight into the regulatory mechanism of lignin synthesis under abiotic stress.