Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in ...several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.
Summary
The secondary cell wall is an important carbon sink in higher plants and its biosynthesis requires coordination of metabolic fluxes in the phenylpropanoid pathway. In Arabidopsis (Arabidopsis ...thaliana), MYB75 and the KNOX transcription factor KNAT7 form functional complexes to regulate secondary cell wall formation in the inflorescence stem. However, the molecular mechanism by which these transcription factors control different branches of the phenylpropanoid pathway remains poorly understood in woody species. We isolated an R2R3‐MYB transcription factor MYB6 from Populus tomentosa and determined that it was expressed predominately in young leaves. Overexpression of MYB6 in transgenic poplar upregulated flavonoid biosynthetic gene expression, resulting in significantly increased accumulation of anthocyanin and proanthocyanidins. MYB6‐overexpression plants showed reduced secondary cell wall deposition, accompanied by repressed expression of secondary cell wall biosynthetic genes. We further showed that MYB6 interacted physically with KNAT7 and formed functional complexes that acted to repress secondary cell wall development in poplar and Arabidopsis. The results provide an insight into the transcriptional mechanisms involved in the regulation of the metabolic fluxes between the flavonoid and lignin biosynthetic pathways in poplar.
Significance Statement
The poplar MYB6 transcription factor is involved in the positive regulation of anthocyanin and proanthocyanidin biosynthesis but the inhibition of secondary cell wall formation with KNAT7 repressor.
Key message
A novel R2R3 MYB transcription factor
PtrMYB57
interacted with bHLH131 and PtrTTG1 to form the MBW complex and negatively regulated the biosynthesis of both anthocyanins and PAs in ...poplar.
R2R3-MYB transcription factors (TFs) are important regulators of secondary metabolite biosynthesis in woody species. A series of R2R3-MYB TFs involved in anthocyanin and proanthocyanidin (PA) biosynthesis have been identified in poplar. In this study, we report the identification and characterization of a subgroup 4 MYB member PtrMYB57, which contains a repressor domain (LxLxL) at the C-terminal end.
PtrMYB57
encodes an R2R3 MYB protein localized in the nucleus and is predominantly expressed in mature leaves. Transgenic poplar overexpressing
PtrMYB57
showed a reduction in anthocyanin and PA accumulation compared to wild-type plants. By contrast, a high anthocyanin and PA phenotype was observed in
Ptrmyb57
mutants generated by the CRISPR/Cas9 system. Furthermore, transient expression assays revealed that PtrMYB57 interacted with bHLH131 (bHLH) and PtrTTG1 (WDR) to form the MBW complex and bound to the flavonoid gene promoters, leading to inhibition of these promoters. Taken together, our results suggest that PtrMYB57 plays a negative role in the regulation of anthocyanin and PA biosynthesis in poplar.
Salt-induced developmental plasticity in a plant root system strongly depends on auxin signaling. However, the molecular events underlying this process are poorly understood. MicroRNA390 (miR390), ...trans-actin small interfering RNAs (tasiRNAs), and AUXIN RESPONSE FACTORs (ARFs) form a regulatory module involved in controlling lateral root (LR) growth. Here, we found that miR390 expression was strongly induced by exposure to salt during LR formation in poplar (Populus spp.) plants. miR390 overexpression stimulated LR development and increased salt tolerance, whereas miR390 knockdown caused by a short tandem target mimic repressed LR growth and compromised salt resistance. ARF3.1, ARF3.2, and ARF4 expression was inhibited significantly by the presence of salt, and transcript abundance was decreased dramatically in the miR390-overexpressing line but increased in the miR390-knockdown line. Constitutive expression of ARF4m harboring mutated trans-acting small interfering ARF-binding sites removed the salt resistance of the miR390 overexpressors. miR390 positively regulated auxin signaling in LRs subjected to salt, but ARF4 inhibited auxin signaling. Salinity stabilized the poplar Aux/IAA repressor INDOLE-3-ACETIC ACID17.1, and overexpression of an auxin/salt-resistant form of this repressor suppressed LR growth in miR390-overexpressing and ARF4-RNA interfering lines in the presence of salt. Thus, the miR390/TAS3/ARFs module is a key regulator, via modulating the auxin pathway, of LR growth in poplar subjected to salt stress.
Proanthocyanidins (PAs) are major defense phenolic compounds in the leaves of poplar (Populus spp.) in response to abiotic and biotic stresses. Transcriptional regulation of PA biosynthetic genes by ...the MYB-basic helix–loop–helix (bHLH)-WD40 complexes in poplar is not still fully understood.
Here, an Arabidopsis TT2-like gene MYB115 was isolated from Populus tomentosa and characterized by various molecular, genetic and biochemical approaches.
MYB115 restored PA productions in the seed coat of the Arabidopsis tt2 mutant. Overexpression of MYB115 in poplar activated expression of PA biosynthetic genes, resulting in a significant increase in PA concentrations. By contrast, the CRISPR/Cas9-generated myb115 mutant exhibited reduced PA content and decreased expression of PA biosynthetic genes. MYB115 directly activated the promoters of PA-specific structural genes. MYB115 interacted with poplar TT8. Coexpression of MYB115, TT8 and poplar TTG1 significantly enhanced the expression of ANR1 and LAR3. Additionally, transgenic plants overexpressing MYB115 had increased resistance to the fungal pathogen Dothiorella gregaria, whereas myb115 mutant exhibited greater sensitivity compared with wild-type plants.
Our data provide insight into the regulatory mechanisms controlling PA biosynthesis by MYB115 in poplar, which could be effectively employed for metabolic engineering of PAs to improve resistance to fungal pathogens.
Isoflavonoids, the major secondary metabolites within the flavonoid biosynthetic pathway, play important roles in plant defense and exhibit free radical scavenging properties in mammals. Recent ...advancements in understanding the synthesis, transport, and regulation of isoflavonoids have identified their biosynthetic pathways as promising targets for metabolic engineering, offering potential benefits such as enhanced plant resistance, improved biomass, and restoration of soil fertility. This review provides an overview of recent breakthroughs in isoflavonoid biosynthesis, encompassing key enzymes in the biosynthetic pathway, transporters influencing their subcellular localization, molecular mechanisms regulating the metabolic pathway (including transcriptional and post-transcriptional regulation, as well as epigenetic modifications). Metabolic engineering strategies aimed at boosting isoflavonoid content in both leguminous and non-leguminous plants. Additionally, we discuss emerging technologies and resources for precise isoflavonoid regulation. This comprehensive review primarily focuses on model plants and crops, offering insights for more effective and sustainable metabolic engineering approaches to enhance nutritional quality and stress tolerance.
Ionic aluminum (Al) in acidic soils, comprising approximately 50% of arable land globally, is highly toxic to most plant species. Populus grow naturally in acidic soils and tolerate high ...concentrations of Al. Multidrug and toxic compound extrusion (MATE) family genes in plants are involved in responses to Al tolerance. To date, however, the functional roles of the MATE genes in Populus remain unclear. In the present study, 71 putative MATE transporters were predicted in the genome of Populus trichocarpa. The chromosome distribution, phylogenetic relationships, and expression level analysis revealed that four candidate MATE genes belonging to subgroup IIIc might contribute to high Al tolerance in poplar. Further, the expression levels of two subgroup IIIc members, PtrMATE1 and PtrMATE2, were induced by Al stress. Transient expression in onion epidermal cells showed that PtrMATE1 was localized to the plasma membrane. Overexpression of PtrMATE1 increased Al-induced secretion of citrate from the root apex of transgenic plants. Al-induced inhibition of root growths were alleviated in both PtrMATE1 overexpression lines in Populus and in Arabidopsis compared with wild-type plants. In addition, PtrMATE1 expression was induced at 12 h after exposure to Al stress whereas PtrMATE2 expression was induced at 24 h, indicating that these proteins coordinately function in response to Al stress in poplar. Taken together, these results provide important insights into the molecular mechanisms involved in Al tolerance in poplar.
Main conclusion
The mechanisms underlying long-term complete submergence tolerance in
S. variegata
involve enhanced oxidative stress responses, strengthened ethylene and ABA signaling, synthesis ...of raffinose family oligosaccharides, unsaturated fatty acids, and specific stress-related amino acids.
Salix variegata
Franch. is a riparian shrub species that can tolerate long-term complete submergence; however, the molecular mechanisms underlying this trait remain to be elucidated. In this study, we subjected
S. variegata
plants to complete submergence for 60 d and collected stems to perform transcriptomic and metabolomic analyses, as well as quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. Results revealed that photosynthesis and the response to light stimulus were inhibited during submergence and recovered after desubmergence. Ethylene and abscisic acid (ABA) signaling could be important for the long-term submergence tolerance of
S. variegata
. Jasmonic acid (JA) signaling also participated in the response to submergence. Raffinose family oligosaccharides, highly unsaturated fatty acids, and specific stress-related amino acids accumulated in response to submergence, indicating that they may protect plants from submergence damage, as they do in response to other abiotic stressors. Several organic acids were produced in
S. variegata
plants after submergence, which may facilitate coping with the toxicity induced by submergence. After long-term submergence, cell wall reorganization and phenylpropanoid metabolic processes (the synthesis of specific phenolics and flavonoids) were activated, which may contribute to long-term
S. variegata
submergence tolerance; however, the detailed mechanisms require further investigation. Several transcription factors (TFs), such as MYB, continuously responded to submergence, indicating that they may play important roles in the responses and adaption to submergence. Genes related to oxidative stress tolerance were specifically expressed after desubmergence, potentially contributing to recovery of
S. variegata
plants within a short period of time.
Because of the importance of wood in many industrial applications, tremendous studies have been performed on wood formation, especially in lignin biosynthesis. MYB transcription factors (TFs), which ...consist of a large family of plant TFs, have been reported to directly regulate lignin biosynthetic genes in a number of plants. In this study, we describe the cloning and functional characterization of PtoMYB216, a cDNA isolated from Chinese white poplar (Populus tomentosa Carr.). PtoMYB216 encodes a protein belonging to the R2R3-MYB family and displays significant similarity with other MYB factors shown to regulate lignin synthesis in Arabidopsis. Gene expression profiling studies showed that PtoMYB216 mRNA is specifically expressed during secondary wall formation in wood. The 1.8-kb promoter sequence of PtoMYB216 was fused to the GUS coding sequence and introduced into wild-type A. thaliana. GUS expression was shown to be restricted to tissues undergoing secondary cell wall formation. Overexpression of PtoMYB216 specifically activated the expression of the upstream genes in the lignin biosynthetic pathway and resulted in ectopic deposition of lignin in cells that are normally unligninified. These results suggest that PtoMYB216 is specific transcriptional activators of lignin biosynthesis and involved in the regulation of wood formation in poplar.
WRKY transcription factors (WRKY TFs) are one of the largest protein families in plants, and most of them play vital roles in response to biotic and abiotic stresses by regulating related signaling ...pathways. In this study, we isolated two WRKY TF genes
and
from
and overexpressed them in Arabidopsis. Expression pattern analyses showed that
and
respond to salicylic acid (SA), methyl JA (MeJA), abscisic acid (ABA),
, and
treatment. The transgenic plants conferred higher
tolerance than wild-type (WT) plants, and real-time quantitative (qRT)-PCR assays showed that
and
had higher expression levels in transgenic plants, which was consistent with their tolerance to
The transgenic plants showed lower
tolerance than WT plants, and qRT-PCR analysis (
,
, and
) also corresponded to this phenotype. Germination rate and root analysis showed that the transgenic plants are less sensitive to ABA, which leads to the reduced tolerance to osmotic stress and the increase of the death ratio and stomatal aperture. Compared with WT plants, a series of ABA-related genes
,
,
,
, and
) were significantly down-regulated in
and
overexpression plants. All of these results demonstrated that the two WRKY TFs are multifunctional transcription factors in plant resistance.
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