Activity of the vascular cambium gives rise to secondary xylem for wood formation in trees. The transcription factor WUSCHEL-related HOMEOBOX4 (WOX4) is a central regulator downstream of the hormone and peptide signaling pathways that maintain cambial activity. However, the genetic regulatory network underlying WOX4-mediated wood formation at the post-transcriptional level remains to be elucidated. In this study, we identified the ubiquitin receptor PagDA1 in hybrid poplar (Populus alba × Populus glandulosa clone 84K) as a negative regulator of wood formation, which restricts cambial activity during secondary growth. Overexpression of PagDA1 in poplar resulted in a relatively reduced xylem due to decreased cambial cell division. By contrast, mutation of PagDA1 by CRISPR/Cas9 resulted in an increased cambial cell activity and promoted xylem formation. Genetic analysis demonstrated that PagDA1 functions antagonistically in a common pathway as PagWOX4 to regulate cambial activity. We propose that PagDA1 physically associates with PagWOX4 and modulates the degradation of PagWOX4 by the 26S proteasome. Moreover, genetic analysis revealed that PagDA1 exerts its negative effect on cambial development by modulating the stability of PagWOX4 in a ubiquitin-dependent manner mediated by the E3 ubiquitin ligase PagDA2. In sum, we have identified a cambial regulatory protein complex, PagDA1–PagWOX4, as a potential target for wood biomass improvement.

The GRAS family genes encode plant-specific transcription factors that play important roles in a diverse range of developmental processes and abiotic stress responses. However, the information of GRAS gene family in the bioenergy crop Miscanthus has not been available. Here, we report the genome-wide identification of GRAS gene family in Micanthus sinensis. A total of 123 MsGRAS genes were identified, which were divided into ten subfamilies based on the phylogenetic analysis. The co-linearity analysis revealed that 59 MsGRAS genes experienced segmental duplication, forming 35 paralogous pairs. The expression of six MsGRAS genes in responding to salt, alkali, and mixed salt-alkali stresses was analyzed by transcriptome and real-time quantitative PCR (RT-qPCR) assays. Furthermore, the role of MsGRAS60 in salt and alkali stress response was characterized in transgenic Arabidopsis. The MsGRAS60 overexpression lines exhibited hyposensitivity to abscisic acid (ABA) treatment and resulted in compromised tolerance to salt and alkali stresses, suggesting that MsGRAS60 is a negative regulator of salt and alkali tolerance via an ABA-dependent signaling pathway. The salt and alkali stress-inducible MsGRAS genes identified serve as candidates for the improvement of abiotic stress tolerance in Miscanthus.

The homeodomain-leucine zipper (HD-ZIP) transcription factors, representing one of the largest plant-specific superfamilies, play important roles in the response to various abiotic stresses. However, the functional roles of HD-ZIPs in abiotic stress tolerance and the underlying mechanisms remain relatively limited in Miscanthus sinensis. In this study, we isolated an HD-ZIP TF gene, MsHDZ23, from Miscanthus and ectopically expressed it in Arabidopsis. Transcriptome and promoter analyses revealed that MsHDZ23 responded to salt, alkali, and drought treatments. The overexpression (OE) of MsHDZ23 in Arabidopsis conferred higher tolerance to salt and alkali stresses compared to wild-type (WT) plants. Moreover, MsHDZ23 was able to restore the hb7 mutant, the ortholog of MsHDZ23 in Arabidopsis, to the WT phenotype. Furthermore, MsHDZ23-OE lines exhibited significantly enhanced drought stress tolerance, as evidenced by higher survival rates and lower water loss rates compared to WT. The improved drought tolerance may be attributed to the significantly smaller stomatal aperture in MsHDZ23-OE lines compared to WT. Furthermore, the accumulation of the malondialdehyde (MDA) under abiotic stresses was significantly decreased, accompanied by dramatically enhanced activities in several antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic plants. Collectively, these results demonstrate that MsHDZ23 functions as a multifunctional transcription factor in enhancing plant resistance to abiotic stresses.

Biochar has been widely reported to improve soil conditions and affect plant growth. However, its effectiveness is limited by soil type and production technology. Considering the application effect of biochar in saline alkali soil, there is currently a lack of in-depth mechanism explanations in the research. Therefore, we designed an experiment to explore the effect of biochar on plant growth in saline alkali soil and conducted soil column experiments in a greenhouse environment using composite inorganic fertilizer (NPK). The results showed that biochar significantly affected the distribution of soil nutrient content at different depths, with a significant increase in fertility levels in the surface and middle layers and a decrease in fertility levels in deep soils. Compared to using fertilizers alone, the combined use of biochar and fertilizers further expands the enrichment effect and significantly reduces the leaching of fertilizers into deeper layers. At the same time, the application of biochar also improved soil properties, including an increase in electrical conductivity and organic matter content, as well as an increase in soil enzyme activity. On the other hand, the application of biochar also increases the activity of antioxidant enzymes and the content of osmoregulation substances in plants, reducing the environmental stress that plants are subjected to. Therefore, our results indicate that biochar can reduce the leaching of fertilizers into deep soil layers, improve soil properties, and promotes the growth of Miscanthus in saline alkali soils.