Cinnamyl alcohol dehydrogenase (CAD) plays a crucial role in lignin biosynthesis, and the gene family encoding various CAD isozymes has been cloned and characterized in numerous plant species. However, limited information regarding the CAD gene family in tobacco is currently available. In this study, we identified 10 CAD genes in Nicotiana tabacum, four in N. tomentosiformis, and six in N. sylvestris. The nucleotide and amino acid sequences of these tobacco CADs demonstrate high levels of similarity, whereas the putative protein sequences conservatively possessed two Zn2+ binding motifs and an NADP(H) cofactor binding motif. Both NtCAD1 and NtCAD2 had conservative substrate binding sites, similar to those possessed by bona fide CADs, and evidence from phylogenetic analysis as well as expression profiling supported their role as bona fide CADs involved in lignin biosynthesis. NtCAD1 has two paralogous genes, NtCAD1-1 and NtCAD1-2. Enzyme activity analysis revealed that NtCAD1-1 and NtCAD1-2 had a high affinity to coniferyl aldehyde, p-coumaryl aldehyde, and sinapyl aldehyde, whereas NtCAD2 preferred coniferyl aldehyde and p-coumaryl aldehyde as substrates. The kinetic parameter assay revealed that NtCAD1-2 functions as the most efficient enzyme. Downregulation of both NtCAD1-1 and NtCAD1-2 resulted in reddish-brown stems without significant changes in lignin content. Furthermore, NtCAD1-1, NtCAD1-2, and NtCAD2 showed distinct expression patterns in response to biotic and abiotic stresses, as well as different phytohormones. Our findings suggest that NtCAD1-1 and NtCAD1-2 are involved in lignin biosynthesis, with NtCAD1-2 also participating in both biological and abiotic stresses, whereas NtCAD2 plays a distinct role mainly in responding to biological and abiotic stresses in tobacco.

The petals of ornamental plants such as roses (Rosa spp.) are the most economically important organs. This delicate, short-lived plant tissue is highly susceptible to pathogens, in large part because the walls of petal cells are typically thinner and more flexible compared with leaf cells, allowing the petals to fold and bend without breaking. The cell wall is a dynamic structure that rapidly alters its composition in response to pathogen infection, thereby reinforcing its stability and boosting plant resistance against diseases. However, little is known about how dynamic changes in the cell wall contribute to resistance to Botrytis cinerea in rose petals. Here, we show that the B. cinerea-induced transcription factor RhbZIP17 is required for the defense response of rose petals. RhbZIP17 is associated with phenylpropanoid biosynthesis and binds to the promoter of the lignin biosynthesis gene RhCAD1, activating its expression. Lignin content showed a significant increase under gray mold infection compared with the control. RhCAD1 functions in the metabolic regulation of lignin production and, consequently, disease resistance, as revealed by transient silencing and overexpression in rose petals. The WRKY transcription factor RhWRKY30 is also required to activate RhCAD1 expression and enhance resistance against B. cinerea. We propose that RhbZIP17 and RhWRKY30 increase lignin biosynthesis, improve the resistance of rose petals to B. cinerea, and regulate RhCAD1 expression.

Shikonin derivatives are natural naphthoquinone compounds and the main bioactive components produced by several boraginaceous plants, such as Lithospermum erythrorhizon and Arnebia euchroma. Phytochemical studies utilizing both L. erythrorhizon and A. euchroma cultured cells indicate the existence of a competing route branching out from the shikonin biosynthetic pathway to shikonofuran. A previous study has shown that the branch point is the transformation from (Z)-3\'\'-hydroxy-geranylhydroquinone to an aldehyde intermediate (E)-3\'\'-oxo-geranylhydroquinone. However, the gene encoding the oxidoreductase that catalyzes the branch reaction remains unidentified. In this study, we discovered a candidate gene belonging to the cinnamyl alcohol dehydrogenase family, AeHGO, through coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines of A. euchroma. In biochemical assays, purified AeHGO protein reversibly oxidized (Z)-3\'\'-hydroxy-geranylhydroquinone to produce (E)-3\'\'-oxo-geranylhydroquinone followed by reversibly reducing (E)-3\'\'-oxo-geranylhydroquinone to (E)-3\'\'-hydroxy-geranylhydroquinone, resulting in an equilibrium mixture of the three compounds. Time course analysis and kinetic parameters showed that the reduction of (E)-3\'\'-oxo-geranylhydroquinone was stereoselective and efficient in presence of NADPH, which determined that the overall reaction proceeded from (Z)-3\'\'-hydroxy-geranylhydroquinone to (E)-3\'\'-hydroxy-geranylhydroquinone. Considering that there is a competition between the accumulation of shikonin and shikonofuran derivatives in cultured plant cells, AeHGO is supposed to play an important role in the metabolic regulation of the shikonin biosynthetic pathway. Characterization of AeHGO should help expedite the development of metabolic engineering and synthetic biology toward production of shikonin derivatives.

[Objective] Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. The aim of this study was to identify CAD gene family members in pomegranate and its expression correlation with seed hardness. [Methods] Based on the reported CAD sequence of Arabidopsis, the CAD gene family of pomegranate was identified by homologous comparison, and then phylogenetic, molecular characterization, and expression profile analysis were performed. [Results] Pomegranate CAD gene family has 25 members, distributed on seven chromosomes of pomegranate. All pomegranate CAD proteins have similar physical and chemical properties. We divide the family into four groups based on evolutionary relationships. The member of group I, called bona fide CAD, was involved in lignin synthesis. Most of the members of group II were involved in stress resistance. The functions of groups III and IV need to be explored. We found four duplicated modes (whole genome duplication or segmental (WGD), tandem duplication (TD), dispersed duplication (DSD), proximal duplication (PD) in this family; TD (36%) had the largest number of them. We predicted that 20 cis-acting elements were involved in lignin synthesis, stress resistance, and response to various hormones. Gene expression profiles further demonstrated that the PgCAD gene family had multiple functions. [Conclusions] Pomegranate CAD gene family is involved in lignin synthesis of hard-seeded cultivar Hongyushizi and Baiyushizi, but its role in seed hardness of soft-seeded cultivar Tunisia needs to be further studied.

BACKGROUND: Physcomitrium patens provides an evolutionary link between green algae and vascular plants. Although the genome of P. patens includes orthologs of all the core lignin biosynthetic enzymes, the occurrence of lignin in moss is very controversial. Besides, little information is available about the lignin enzymes in moss to date. For example, cinnamyl alcohol dehydrogenase (CAD) is a crucial enzyme that catalyzes the last step of the lignin biosynthetic pathway, suggesting an ideal way to study the evolutionary process. By investigating the functions of CAD in evolution, this study will elucidate the evolutionary roles of lignin-like in the early stage of land colonization.
RESULTS: CAD multigene family in P. patens is composed of four genes. The PpCADs contain a conserved glycine-rich domain to catalyze NADPH-dependent reduction to their corresponding alcohols, indicating that PpCADs have the potential to synthesize monolignols by bioinformatics analysis. Even though PpCAD1 could produce lignin in theory, no conventional monomer was detected in the cell wall or cytoplasm of PpCAD1_OE plants. However, the phenylpropanoids were promoted in PpCAD1_OE transformants to modify gametophore architecture and development, making the distribution of phyllids more scarcity and the moss colony more giant, possibly due to the enhanced expression of the AUX-IAA family. The transcripts of at least one gene encoding the enzyme in the lignin biosynthetic pathway were increased in PpCAD1_OE plants. In addition, the PpCAD1_OE gametophore inhibited the Botrytis cinerea assault mainly by enhanced phenylpropanoids in the cell wall instead of influencing transcripts of defense genes pathogenesis-related 10 (PR10) and nonexpresser of PR genes 1 (NPR1). Likewise, ectopic expression of PpCAD1 in Arabidopsis led to a significant increase in lignin content, exhibiting chunky roots, robust seedlings, advanced flowering, and efficient resistance against pathogens.
CONCLUSIONS: PpCAD occurs in more than one copy, suggesting functional divergence in the ancestral plant. PpCAD1 catalyzes monolignol biosynthesis and has homologous functions with vascular plants. Despite no detected conventional monolignol, the increased phenylpropanoids in the PpCAD1_OE gametophore, possibly intermediate metabolites in the lignin pathway, had conserved functions during the evolution of terrestrial plants. The results inferred that the lignin enzyme of the early non-vascular plant played roles in stem elongation and resistance against pathogens of P. patens during the conquest of land.

Mulberry (Morus) is used as a feed additive and biofuel materials. Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95) catalyzes the final step of monolignol biosynthesis and is responsible for various monolignols. Five MaCADs from Morus alba were cloned and functionally characterized in the present study. These MaCADs encoded proteins with 357-364 amino acids, and the putative protein sequences conservatively possessed two Zn2+ binding motifs and an NADP(H) cofactor binding motif. However, MaCAD1, 2, and 5 shared similar amino acids at substrate binding positions that differed from those possessed by bona fide CADs. MaCAD3 and 4 had conservative substrate binding sites, and both phylogenetic and expression profile analysis indicated they were bona fide CADs involved in lignin biosynthesis. The enzymatic assay showed that MaCAD1 and 5 had a high affinity to p-coumaryl aldehyde. MaCAD4 preferentially used coniferyl aldehyde and sinapyl aldehyde as substrates. His-72 and Tyr-124 in MaCAD1 stabilized p-coumaryl aldehyde, and may have resulted in the substrate preference for p-coumaryl aldehyde. Down-regulation of MaCADs in mulberry showed that MaCAD3/4 were dominant CADs that functioned in monolignol biosynthesis, and decreased MaCAD3/4 resulted in significant decreases of lignin content in both stems and leaves. MaCADs exhibited different expression patterns in response to various stresses, indicating their possible diverse roles. MaCAD2 and MaCAD5 may play positive roles in response to drought and cold stresses, respectively. These results provide a systematic functional analysis of MaCADs in mulberry and an important foundation for the genetic modification of the monolignol pathway in mulberry.

Cinnamyl alcohol dehydrogenase (CAD) is the key enzyme for lignin biosynthesis in plants. In this study, genome-wide analysis was performed to identify CAD genes in oil palm (Elaeis guineensis). Phylogenetic analysis was then conducted to select the bona fide EgCADs. The bona fide EgCAD genes and their respective 5\' flanking regions were cloned and analysed. Their expression profiles were evaluated in various organs using RT-PCR. Seven EgCAD genes (EgCAD1-7) were identified and divided into four phylogenetic groups. EgCAD1 and EgCAD2 display high sequence similarities with other bona fide CADs and possess all the signature motifs of the bona fide CAD. They also display similar 3D protein structures. Gene expression analysis showed that EgCAD1 was expressed most abundantly in the root tissues, while EgCAD2 was expressed constitutively in all the tissues studied. EgCAD1 possesses only one transcription start site, while EgCAD2 has five. Interestingly, a TC microsatellite was found in the 5\' flanking region of EgCAD2. The 5\' flanking regions of EgCAD1 and EgCAD2 contain lignin-associated regulatory elements i.e. AC-elements, and other defence-related motifs, including W-box, GT-1 motif and CGTCA-motif. Altogether, these results imply that EgCAD1 and EgCAD2 are bona fide CAD involved in lignin biosynthesis during the normal development of oil palm and in response to stresses. Our findings shed some light on the roles of the bona fide CAD genes in oil palm and pave the way for manipulating lignin content in oil palm through a genetic approach.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s13205-022-03208-0.

Floral scent is an important economic and ornamental trait of Prunus mume. The floral volatiles from most cultivars of P. mume in composition exist significant differences. Cinnamyl alcohol was one of the main floral volatile compounds with distinct abundances in different cultivars, namely, \'Zaohua Lve,\' \'Zao Yudie,\' \'Fenpi Gongfen,\' \'Jiangsha Gongfen,\' and \'Fenhong Zhusha.\' Based on the determination of endogenous volatiles of full-blooming flowers, vital enzyme activity and transcriptomes were comprehensively analyzed to screen the key potential genes involved in cinnamyl alcohol synthesis. Transcriptome combining with enzyme activity level analysis suggested that the expression levels of three PmCADs were highly correlated with the cinnamyl alcohol dehydrogenase (CAD) enzyme activities in six cultivars. Furthermore, phylogenetic tree and transcriptome analysis suggested that PmCAD1 and PmCAD2 might contribute to the cinnamyl alcohol synthesis. Relative expression analyses and enzyme activity assays showed that PmCAD1 played an important role in cinnamyl alcohol biosynthesis in vitro. Overall, this research lays a theoretical foundation for clarifying comprehensively the molecular biosynthesis mechanism of floral volatiles in P. mume.

Tall fescue, a promising temperate forage grass of Himalayan region, possesses extraordinary property of rapid growth with high biomass production, but its poor digestibility due to higher lignin content limits its utilization in livestock feeding. The lignification in Tall fescue is under the control of enzymatic cascade of different regulatory enzymes. Cinnamyl alcohol dehydrogenase (CAD) is a crucial regulatory enzyme that catalyzes the last step of monolignol biosynthesis and is a potential candidate for altering the content and types of lignin, and hence increasing the digestibility of fodder crops. Hence, the present investigation was conducted on isolation, cloning and characterization of CAD gene from Tall fescue. Isolation and amplification of CAD gene resulted in an amplicon of 1521 bp. The CAD gene sequence was submitted to NCBI database with an accession number MW442831. Translation of the CAD gene sequence exhibited an ORF of 361 amino acids. The deduced CAD protein was predicted to be hydrophobic, acidic and thermally stable with molecular formula C1712H2734N460O520S23, molecular mass of 38.82 kDa, theoretical pI of 5.60 and 3 strong transmembrane helices. The CAD protein was predicted to have a dimer forming behavior with putative NAD(P) binding site between amino acids 48 and 301, putative substrate-binding site between amino acids 48 and 301, catalytic zinc-binding site between amino acids 48 and 164 and structural zinc-binding site between amino acid residue 101 and 115. A conserved 189GLGGVG194 motif is the binding site for NADP(H). The conserved motif pattern of CAD\'s zinc catalytic center was found to be 69GHEVVGEV(X)EVG(X)2V83. The zinc-binding site was found to be conserved between amino acid 89 and 115 and was found to be 89G(X)2VG(X)G(X)2VGXC(X)2C(X)2C(X)5QYC115. The deciphered sequence and putative protein information might be useful in subsequent research in lignin bioengineering for enhanced digestibility, biomass conversion as well as impact of lignin on cell wall mechanics.

The synthetic enzyme cinnamyl alcohol dehydrogenase (CAD) is involved in responses to various stresses during plant growth. It regulates the monolignol biosynthesis and catalyzes hydroxyl cinnamaldehyde reduction to the corresponding alcohols. Although the CAD gene families have been explored in some species, little known is in Rosaceae. In this study, we identified 149 genes in Pyrus bretschneideri (PbrCAD), Malus domestica (MDPCAD), Prunus mume (PmCAD) and Fragaria vesca (mrnaCAD). They were phylogenetically clustered into six subgroups. All CAD genes contained ADH-N and ADH-zinc-N domains and were distributed on chromosomes unevenly. Dispersed and WGD/segmental duplications accounted the highest number of evolutionary events. Eight collinear gene pairs were identified among the four Rosaceae species, and the highest number was recorded in pear as five pairs. The five PbrCAD gene pairs had undergone purifying selection under Ka/Ks analysis. Furthermore, nine genes were identified based on transcriptomic and stone cell content in pear fruit. In qRT-PCR, the expression patterns of PbrCAD1, PbrCAD20, PbrCAD27, and PbrCAD31 were consistent with variation in stone cell content during pear fruit development. These results will provide valuable information for understanding the relationship between gene expressions and stone cell number in fruit.