Drought stress severely affects global plant growth and production. The enhancement of plant water-use efficiency (WUE) and drought tolerance by the manipulation of the stomata is an effective strategy to deal with water shortage. However, increasing the WUE and drought tolerance by manipulation on the stomata has rarely been tested in Brassica napus. Here, we isolated Bna.EPF2, an epidermal patterning factor (EPF) in Brassica napus (ecotype Westar), and identified its role in drought performance. Bna.EPF2 overexpression lines had a reduction average of 19.02% in abaxial stomatal density and smaller stomatal pore size, leading to approximately 25% lower transpiration, which finally resulted in greater instantaneous WUE and enhanced drought tolerance. Interestingly, the reduction in stomatal density did not affect the CO₂ assimilation or yield-related agronomic traits in Bna.EPF2 overexpression plants. Together with the complementation of Bna.EPF2 significantly decreasing the stomatal density of Arabidopsis epf2, and Bna.EPF2 being expressed in mature guard cells, these results suggest that Bna.EPF2 not only functions in stomatal density development, but also in stomatal dimension in Brassicas. Taken together, our results suggest that Bna.EPF2 improves WUE and drought tolerance by the regulation of stomatal density and stomatal size in Brassica without growth and yield penalty, and provide insight into the manipulation of this gene in the breeding of drought tolerant plants with increased production under water deficit conditions.

Drought stress seriously threatens plant growth. The improvement of plant water use efficiency (WUE) and drought tolerance through stomatal regulation is an effective strategy for coping with water shortages. Epidermal patterning factor (EPF)/EPF-like (EPFL) family proteins regulate stomatal formation and development in plants and thus contribute to plant stress adaptation. Here, our analysis revealed the presence of 14 PeEPF members in the Populus euphratica genome, which exhibited a relatively conserved gene structure with 1–3 introns. Subcellular localisation prediction revealed that 9 PeEPF members were distributed in the chloroplasts of P. euphratica, and 5 were located extracellularly. Phylogenetic analysis indicated that PeEPFs can be divided into three clades, with genes within the same clade revealing a relatively conserved structure. Furthermore, we observed the evolutionary conservation of PeEPFs and AtEPF/EPFLs in certain domains, which suggests their conserved function. The analysis of cis-acting elements suggested the possible involvement of PeEPFs in plant response to multiple hormones. Transcriptomic analysis revealed considerable changes in the expression level of PeEPFs during treatment with polyethylene glycol and abscisic acid. The overexpression of PeEPF2 resulted in low stomatal density in transgenetic lines. These findings provide a basis for gaining insights into the function of PeEPFs in response to abiotic stress.

Populus euphratica Oliv. typically has four kinds of heteromorphic leaves: linear (Li), lanceolate (La), ovate (Ov) and broad ovate (Bo). Heteromorphic leaves help P. euphratica adapt to extreme desert environments and further contribute to protection against land desertification in Northwest China. In the authors’ previous research, growth-regulating factors (GRFs) were speculated to be related to the development of P. euphratica heteromorphic leaves via multi-omics integrated analysis. However, the genomic features and biological role of the P. euphratica GRF gene family in heteromorphic leaves are still unclear. In this study, 19 PeGRF genes were genome-widely identified and characterized in P. euphratica, and their physicochemical properties, gene structure and phylogenetic evolution were analyzed. An analysis of the research showed that PeGRFs were unevenly distributed on 11 chromosomes and that PeGRF proteins contained conserved motif 1 (WRC) and motif 2 (QLQ). Moreover, 19, 15, 19 and 22 GRFs were identified in Populus deltoides Marshall, Populus pruinosa Schrenk, Salix sinopurpurea C. Wang et C. Y. Yang and Salix suchowensis W. C. Cheng, respectively. A collinearity analysis showed that the PeGRF family evolved slowly within Populus species. A phylogenetic tree of the GRF family was also constructed, and GRFs were divided into four subfamilies. A large number of cis-acting elements were related to plant growth and development, plant hormone response and stress response on the promoter of PeGRFs. The expression pattern of PeGRFs showed significant up-regulation in broad leaves (Ov and Bo) compared with narrow leaves (Li and La). In combination with the predicted gene regulatory network, PeGRF9 (PeuTF06G01147.1) may have an important contribution to the leaf shape development of P. euphratica. The heterologous expression of PeGRF9 in wild-type plants (Col-0) of Arabidopsis thaliana (L.) Heynh was also studied, showing a significant increase in the leaf area of overexpressing plants compared with the wild type. Nineteen PeGRF gene members were identified and characterized in P. euphratica, and a comparison of the genomic analysis of Populus GRF members revealed their evolutionary features. The further overexpression of PeGRF9 in A. thaliana revealed its biological role in the heteromorphic leaves of P. euphratica. This study not only provides new insights into the evolution and function of PeGRFs in P. euphratica heteromorphic leaves but also helps in an understanding of the adaptive evolution of P. euphratica in drought desert environments.

The basic helix-loop-helix transcription factor SCREAM (SCRM), also known as INDUCER OF CBF EXPRESSION 1 (ICE1), functions as a regulatory hub that integrates environmental and hormonal signals to mediate diverse aspects of plant development and stress adaptation. Initially characterized for its roles in cold tolerance and stomatal development, recent studies have expanded its functional landscape to include reproductive development, seed dormancy, leaf senescence, and immune modulation. This review synthesizes current knowledge of SCRM/ICE1’s multifaceted functions across the plant life cycle, summarizing its molecular regulatory networks in development and stress responses respectively, and highlighting its central role as a regulatory node. By consolidating these dispersed findings, we discuss the balance between developmental programming and stress adaptation mediated by SCRM/ICE1, identify key questions for future research, and explore its potential applications in crop genetic improvement.

DNA-binding with one finger (Dof) transcription factors plays critical roles in regulating plant growth and development, as well as modulating responses to biotic and abiotic stresses. While the biological characteristics of the Dof family have been explored across various species, their functions in Populus euphratica remain largely uncharacterized. In this study, we identified 43 PeDof family genes through a genome-wide approach, revealing a total of 10 conserved motifs across all family members. Predictions of cis-acting elements indicated that Dof genes are involved in light signaling, hormone signaling, and stress responses. Phylogenetic analysis classified the 43 Dof genes of P. euphratica into six distinct groups, with genes within the same group exhibiting relatively conserved structures. Expression pattern analyses demonstrated significant regulation of PeDof genes by drought stress, with their expression also being influenced by environmental conditions during seed germination. Furthermore, we identified the Dof gene PeSCAP1, which plays a conserved role in regulating guard cell maturation, underscoring the importance of stomatal morphology and function in leaf water retention. This study enhances our understanding of the role of Dofs in abiotic stress responses and provides valuable insights into their function in Populus euphratica.

Introduction: Xinjiang is one of the important origins of walnut ( Juglans regia L.) and harbors abundant germplasm resources. However, a standardized phenological description system for precocious walnuts in this region is lacking. This study aimed to establish such a system based on the extended BBCH scale. Methods: We conducted systematic phenological observations over two growing seasons (2023 -2024) on six nine-year-old precocious walnut materials (two main cultivars and four elite strains) planted in Alar, Xinjiang. The study covered eight principal growth stages: bud development (0), leaf development (1), shoot development (3), inflorescence emergence (5), flowering (6), fruit development (7), fruit maturity (8), and senescence and dormancy (9), further subdivided into 38 secondary stages, with coded morphological photographs provided for each stage. Results: Significant differences were observed among varieties and elite strains. BT-2 had the earliest budburst, the highest number of female flowers, and reached standard fruit size first; XW-185 had abundant male flowers and could serve as a suitable pollen donor; BT-1 exhibited the highest kernel percentage and the thinnest nutshell. Correlation analysis revealed that the trait correlation patterns of BT-2 and BT-3 were relatively complex, showing opposite trends. PCA and clustering heatmap indicated clear differentiation among the six materials, classifying them into four clusters. Discussion: The BBCH_based system provides a practical tool for pollinizer arrangement, irrigation scheduling, and breeding of precocious walnuts. Complementary traits among varieties support targeted utilization, and phenological stages can guide stage_specific irrigation. However, the two_year observation period limits the assessment of long_term stability, and future research should further elucidate the interaction network between early fruiting traits and quality traits.