Stomata regulate CO₂ and water vapor exchange between leaves and the atmosphere, serving as a vital indicator of climate change resilience. Therefore, understanding the difference in stomatal numbers and patterns among different soybean cultivars across growth stages is essential to comprehending the complex mechanisms underlying soybean adaptation to climate change. The accurate measurements of stomatal density in soybean leaves are essential to understanding the complexity of stomatal density by environmental conditions. We demonstrated that the five epidermal sections and five microscopic images taken from both sides of each epidermal section at each leaf position (tip, middle, and bottom) were sufficient for stomatal measurements. Furthermore, we investigated variations in stomatal density among leaflet locations (left, right, and central) and leaf position across different growth stages. Notably, while there was no significant variation between the two leaves of the vegetative cotyledon (VC) stage and among the three leaflets of the V1 (first trifoliate) to V4 (fourth trifoliate) growth stages, leaves of the VC stage exhibited the lowest stomatal density, whereas those of the V4 stage exhibited the highest stomatal density. These findings could serve as a valuable tool for evaluating stomatal density, analyzing physiological differences under adverse climatic conditions, and phenotyping a large-scale population to identify the genetic factors responsible for stomatal density variations in soybean genotypes.

Plant grafting is a key horticultural practice used to promote agricultural performance by improving crop productivity and enhancing resistance to biotic and abiotic stresses. Successful graft union formation depends on tissue adhesion, callus formation, and vascular reconnection, which are regulated primarily by auxin signaling. Although environmental factors influence grafting union formation by modulating hormonal signaling, the role of photoperiod in graft union formation remains poorly understood. Here, we demonstrate that short-day (SD) photoperiods markedly enhance grafting success in Arabidopsis thaliana by promoting hypocotyl elongation and increasing auxin accumulation at the graft junction. DR5:GUS reporter assays revealed strong photoperiod-dependent differences in auxin responsiveness, with the highest auxin levels under SD conditions and substantially reduced levels under long-day (LD) and medium-day (MD) conditions. Exogenous indole-3-acetic acid (IAA) treatment restored the grafting success rate under LD and MD conditions, whereas inhibition of polar auxin transport by 2,3,5-triiodobenzoic acid (TIBA) significantly reduced grafting success under SD conditions. Notably, TIBA treatment did not lead to a further significant reduction under LD and MD conditions, where endogenous auxin levels were already limiting, confirming the necessity of localized auxin accumulation at the graft junction. Furthermore, transferring grafted seedlings from LD to SD conditions restored grafting success, highlighting the plasticity of light-responsive transcriptional pathways that regulate auxin biosynthesis. Together, these findings reveal that photoperiod enhances grafting success primarily by increasing endogenous auxin accumulation, which in turn enables efficient auxin transport and the activation of auxin-mediated regenerative programs at the graft junction. This work establishes photoperiod as a key environmental determinant of grafting efficiency and provides a framework for optimizing grafting outcomes through the modulation of light conditions and hormonal signaling.

Plant growth habit arises from the integration of developmental regulatory network and environmental signaling pathways. In soybean, the erect growth habit of cultivated soybean (Glycine max) contrasts with the vine growth habit of wild soybean (Glycine soja), the molecular mechanisms linking environmental signal with hormonal regulation of stem elongation remain poorly understood. Here, we investigated the genetic, transcriptional, and hormonal basis of growth habit difference between Williams 82 (erect growth habit) and PI 366121 (vine growth habit). PI 366121 exhibited an acceleration of stem elongation at the V3 growth stage, indicating a developmentally regulated onset of the vine growth habit. QTL mapping using a high-density bin-based linkage map identified a major QTL on chromosome 14, encompassing twenty candidate genes. At V3 growth stage, Williams 82 exhibited transcriptional expression patterns indicative of strengthened photodamage repair and ROS detoxification, whereas PI 366121 exhibited coordinated activation of photoprotective, circadian, and gibberellin-responsive regulatory networks. Weighted gene co-expression network analysis identified an ELIP-mediated regulatory module correlated with the vine growth habit, co-expression of ELIP and GASA22 genes supports functional integration between chloroplast redox homeostasis and gibberellin-mediated stem elongation. We propose a molecular mechanism in which ELIP-mediated photoprotection stabilizes photosystem II redox homeostasis, suppresses CBF-dependent GA2ox gene expression, and sustains bioactive GA accumulation, promoting GASA22 gene activation and stem elongation. This ELIP-GASA22 regulatory module provides a molecular basis for how chloroplast-derived environmental signals modulate gibberellin signaling to shape morphological plasticity in wild soybean.

Plants coordinate cell elongation with the daily light/dark cycle, an important adaptive strategy for optimizing growth under changing environmental conditions. However, the transcriptional mechanisms linking light signals to plant developmental growth remain poorly understood. In this study, we identified the GATA25 transcription factor as a regulator of petiole and hypocotyl elongation in response to light and photoperiod. Overexpression of GATA25 in Arabidopsis thaliana increased petiole and hypocotyl lengths and elevated TCP4 transcript levels. We demonstrated that GATA25 directly binds to the promoter of TCP4 and activates its transcription using transcriptional activation analysis, a glucocorticoid receptor-based inducible system, chromatin immunoprecipitation, and electrophoretic mobility shift assays. Furthermore, yeast two-hybrid assay and bimolecular fluorescence complementation analyses showed that GATA25 physically interacts with the E3 ubiquitin ligase COP1. Notably, overexpression of GATA25 did not increase TCP4 transcript levels in dark conditions, suggesting that GATA25 activity is suppressed in dark conditions. Based on these findings, we propose a model in which GATA25 is destabilized by COP1 in dark conditions, restricting TCP4 activation to the light conditions. Taken together, these findings provide a framework for understanding how the daily light/dark cycle regulates cell elongation in a photoperiod-dependent manner through the GATA25-TCP4 module.