Lignin is one of the main components of plant cell wall and it is a natural phenolic polymer with high molecular weight, complex composition and structure. Lignin biosynthesis extensively contributes to plant growth, tissue/organ development, lodging resistance and the responses to a variety of biotic and abiotic stresses. In the present review, we systematically introduce the biosynthesis of lignin and its regulation by genetic modification and summarize the main biological functions of lignin in plants and their applications. We hope this review will give an in-depth understanding of the important roles of lignin biosynthesis in various plants’ biological processes and provide a theoretical basis for the genetic improvement of lignin content and composition in energy plants and crops.

https://www.mdpi.com/1422-0067/19/2/335/pdf

Lignins: Biosynthesis and Biological Functions in Plants

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

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Laccases: structure, function, and potential application in water bioremediation

In plants, copper (Cu) acts as essential cofactor of numerous proteins. While the definitive number of these so-called cuproproteins is unknown, they perform central functions in plant cells. As micronutrient, a minimal amount of Cu is needed to ensure cellular functions. However, Cu excess may exert in contrast detrimental effects on plant primary production and even survival. Therefore it is essential for a plant to have a strictly controlled Cu homeostasis, an equilibrium that is both tissue and developmentally influenced. In the current review an overview is presented on the different stages of Cu transport from the soil into the plant and throughout the different plant tissues. Special emphasis is on the Cu-dependent responses mediated by the SPL7 transcription factor, and the crosstalk between this transcriptional regulation and microRNA-mediated suppression of translation of seemingly non-essential cuproproteins. Since Cu is an essential player in electron transport, we also review the recent insights into the molecular mechanisms controlling chloroplastic and mitochondrial Cu transport and homeostasis. We finally highlight the involvement of numerous Cu-proteins and Cu-dependent activities in the properties of one of the major Cu-accumulation sites in plants: the cell wall.

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Copper Trafficking in Plants and Its Implication on Cell Wall Dynamics

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

A strong loss-of-function mutation in RAN1 results in constitution activation of the ethylene response pathway as well as a rosette-lethal phenotype

Plants confront multifarious environmental stresses widely divided into abiotic and biotic stresses, of which heavy metal stress represents one of the most damaging abiotic stresses Heavy metals cause toxicity by targeting crucial molecules and vital processes in the plant cell One of the approaches by which heavy metals act in plants is by over production of reactive oxygen species (ROS) either directly or indirectly Plants act against such overdose of metal in the environment by boosting the defense responses like metal chelation, sequestration into vacuole, regulation of metal intake by transporters, and intensification of antioxidative mechanisms This response shown by plants is the result of intricate signaling networks functioning in the cell in order to transmit the extracellular stimuli into an intracellular response The crucial signaling components involved are calcium signaling, hormone signaling, and mitogen activated protein kinase (MAPK) signaling that are discussed in this review Apart from signaling components other regulators like microRNAs and transcription factors also have a major contribution in regulating heavy metal stress This review demonstrates the key role of MAPKs in synchronously controlling the other signaling components and regulators in metal stress Further, attempts have been made to focus on metal transporters and chelators that are regulated by MAPK signaling

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Traversing the Links between Heavy Metal Stress and Plant Signaling.

Alternative splicing (AS) of pre-mRNAs promotes transcriptome and proteome diversity and plays important roles in a wide range of biological processes. However, the role of AS in maintaining mineral nutrient homeostasis in plants is largely unknown. To clarify this role, we obtained whole transcriptome RNA sequencing data from rice (Oryza sativa) roots grown in the presence or absence of several mineral nutrients (Fe, Zn, Cu, Mn, and P). Our systematic analysis revealed 13,291 alternatively spliced genes, representing ∼53.3% of the multiexon genes in the rice genome. As the overlap between differentially expressed genes and differentially alternatively spliced genes is small, a molecular understanding of the plant’s response to mineral deficiency is limited by analyzing differentially expressed genes alone. We found that the targets of AS are highly nutrient-specific. To verify the role of AS in mineral nutrition, we characterized mutants in genes encoding Ser/Arg (SR) proteins that function in AS. We identified several SR proteins as critical regulators of Zn, Mn, and P nutrition and showed that three SR protein-encoding genes regulate P uptake and remobilization between leaves and shoots of rice, demonstrating that AS has a key role in regulating mineral nutrient homeostasis in rice.

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Alternative Splicing Plays a Critical Role in Maintaining Mineral Nutrient Homeostasis in Rice (Oryza sativa)

Laccases are encoded by a multigene family and widely distributed in plant genomes where they play roles oxidizing monolignols to produce higher-order lignin involved in plant development and stress responses. We identified 30 laccase genes (OsLACs) from rice, which can be divided into five subfamilies, mostly expressed during early development of the endosperm, growing roots, and stems. OsLACs can be induced by hormones, salt, drought, and heavy metals stresses. The expression level of OsLAC10 increased 1200-fold after treatment with 20 μM Cu for 12 h. The laccase activities of OsLAC10 were confirmed in an Escherichia coli expression system. Lignin accumulation increased in the roots of Arabidopsis over-expressing OsLAC10 (OsLAC10-OX) compared to wild-type controls. After growth on 1/2 Murashige and Skoog (MS) medium containing toxic levels of Cu for seven days, roots of the OsLAC10-OX lines were significantly longer than those of the wild type. Compared to control plants, the Cu concentration decreased significantly in roots of the OsLAC10-OX line under hydroponic conditions. These results provided insights into the evolutionary expansion and functional divergence of OsLAC family. In addition, OsLAC10 is likely involved in lignin biosynthesis, and reduces the uptake of Cu into roots required for Arabidopsis to develop tolerance to Cu.

https://www.mdpi.com/1422-0067/18/2/209/pdf

Comprehensive Analysis of Rice Laccase Gene (OsLAC) Family and Ectopic Expression of OsLAC10 Enhances Tolerance to Copper Stress in Arabidopsis.

Widely-spread cadmium (Cd) pollution in the soil threatens both crop production and human health. How plants deal with the excess Cd are largely unknown. To evaluate the molecular mechanism by which plants respond to Cd stress, rice seedlings were treated with two concentrations of Cd and subjected to deep RNA sequencing. Comprehensive RNA-Seq analysis of rice roots under two gradients of Cd treatment revealed 1,169 Cd toxicity-responsive genes. These genes were involved in the reactive oxygen species scavenging system, stress response, cell wall formation, ion transport, and signal transduction. Nine out of 93 predicted long non coding RNAs (lncRNAs) were detected as Cd-responsive lncRNAs due to their high correlation with the Cd stress response. In addition, we analyzed alternative splicing (AS) events under different Cd concentrations. 476 differential alternatively spliced genes with 542 aberrant splicing events were identified. GO analysis indicated that these genes were highly enriched in oxidation reduction and cellular response to chemical stimulus. Real-time qRT-PCR validation analysis strengthened the reliability of our RNA-Seq results. The results suggest that post-transcriptional AS regulation may also be involved in plant responses to high Cd stress.

RNA-Seq Analysis of Rice Roots Reveals the Involvement of Post-Transcriptional Regulation in Response to Cadmium Stress

Induction of lignin biosynthesis is an adaptive response of plants subjected to many abiotic stresses. In this study, we examined the response of lignin biosynthesis to copper (Cu) stress, with a particular focus on the regulatory mechanism. We performed a transcriptomic analysis of rice (Oryza sativa L.) roots, and the microarray data on lignin biosynthesis pathway genes were corroborated by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis. Physiological analyses of rice seedlings treated with Cu(II) sulfate (CuSO4) were used to confirm the relationship between excess Cu and lignin biosynthesis. In addition, we examined the role of hydrogen peroxide (H2O2) in Cu-induced lignin biosynthesis through pretreatments with an NADPH oxidase inhibitor (diphenyleneiodonium, DPI) and a H2O2 scavenger (dimethylthiourea, DMTU). Lignin biosynthesis pathway genes were upregulated under Cu stress. The lignin content of rice roots increased significantly with increasing concentrations and durations of Cu treatment; elevations in root lignin content were correlated with marked inhibitions in root growth. Pretreatments with DPI and DMTU inhibited the activities of Cu-induced lignin polymerization enzymes (peroxidase, POD and laccase, LAC) and lignin accumulation in rice roots. Conversely, exogenous H2O2 increased the root lignin content. Rice roots under Cu stress accumulate lignin through enhanced polymerization of lignin monolignol, a mechanism that requires Cu stress induced H2O2.

Luqing Zheng

Papers

Lignins: Biosynthesis and Biological Functions in Plants

Alternative Splicing Plays a Critical Role in Maintaining Mineral Nutrient Homeostasis in Rice (Oryza sativa)

Comprehensive Analysis of Rice Laccase Gene (OsLAC) Family and Ectopic Expression of OsLAC10 Enhances Tolerance to Copper Stress in Arabidopsis.

RNA-Seq Analysis of Rice Roots Reveals the Involvement of Post-Transcriptional Regulation in Response to Cadmium Stress

Transcriptional and physiological analyses identify a regulatory role for hydrogen peroxide in the lignin biosynthesis of copper-stressed rice roots