Tao Qin

To identify novel regulators of plant drought tolerance, we performed a forward genetic screen using thermal infrared imaging to detect alterations in leaf temperature, a proxy for transpiration. This screen identified AT-rich Interacting Domain Protein 4 (ARID4), a protein involved in chromatin remodeling, as a modulator of transpiration. arid4 loss-of-function mutants exhibited cooler leaves, increased water loss from detached leaves, and heightened susceptibility to drought relative to wild-type plants. Stomata of arid4 mutants were hyposensitive to abscisic acid (ABA), displaying reduced ABA-induced reactive oxygen species (ROS) production and impaired stomatal closure. Transcriptome profiling under drought revealed extensive misregulation of genes involved in stress-responses, redox homeostasis, iron acquisition, and photosynthesis. These findings indicate that ARID4 modulates the expression of key regulatory genes to maintain redox homeostasis and photosynthetic efficiency, thereby conferring drought stress tolerance.

Summary: Research on the functions and molecular mechanisms of long non-coding RNAs (lncRNAs) involved in regulating plant freezing tolerance is still in its infancy. Our previous research work identified that lncRNA DROUGHT INDUCED LNCRNA ( DRIR ) regulates gene expressions in Arabidopsis . However, the underlying molecular mechanism is still unknown. This study demonstrates that lncRNA DRIR regulates plant freezing tolerance by affecting alternative splicing patterns of pre-mRNAs. Through chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS), we identified two DRIR interacting proteins: CP29A and CP29B. We showed that the drir ^D mutant, which exhibits elevated DRIR expression and DRIR overexpression lines showed increased sensitivity to freezing stress, whereas DRIR RNAi lines were more tolerant to the stress. CP29A and CP29B bind to nuclear transcripts and, together with DRIR , regulate pre-mRNA alternative splicing under freezing stress. Notably, DRIR induces the relocalization of CP29A and CP29B to autophagosomes, leading to autophagy-mediated protein degradation. Collectively, our findings elucidate the molecular mechanism by which DRIR influences the autophagy-based degradation of its binding proteins CP29A and CP29B, thereby regulating plant freezing tolerance by altering the alternative splicing patterns of pre-mRNAs, providing novel insights into the functions and mechanisms of lncRNAs in plants adapting to freezing environments.

Leaf color is an important trait for the quality and ornamental value of turfgrass. As an essential component of chlorophyll molecules and related pigments, nitrogen is pivotal for leaf coloration. However, the mechanisms underlying nitrogen‐mediated leaf color regulation in perennial turfgrass species remain unclear. In this study, we demonstrate that mitogen‐activated protein kinase 6 (LpMPK6) regulates leaf color in perennial ryegrass by phosphorylating LpMYBR1. LpMPK6 overexpression (OE) plants showed leaf chlorosis under soil conditions without nutrient supplementation or in low‐nitrogen hydroponic cultivation, accompanied by reduced chlorophyll and nitrogen content. Conversely, LpMPK6 RNAi lines maintained less chlorotic leaves and higher levels of chlorophyll and nitrogen under nitrogen‐deficient conditions. Yeast two‐hybrid, pull‐down, co‐immunoprecipitation, and luciferase complementation imaging indicated that LpMPK6 interacted with transcription factor LpMYBR1. Phenotypic analysis revealed that LpMYBR1 functionally antagonized LpMPK6, with more chlorotic leaves and lower levels of chlorophyll and nitrogen in LpMYBR1 RNAi lines, whereas LpMYBR1 OE plants retained more green leaves under nitrogen‐deficient conditions. DNA affinity purification, yeast one‐hybrid, electrophoretic mobility shift, and dual‐luciferase assays demonstrated that LpMYBR1 bound to the promoter of LpNRT1.5 and activated its transcription. Furthermore, LpNRT1.5 regulated leaf color by mediating NO ₃ ⁻ root‐to‐shoot transport. Notably, LpMPK6 negatively regulated this transport process by phosphorylating LpMYBR1 and suppressing its transactivation of LpNRT1.5 . Taken together, our results revealed a mechanism whereby LpMPK6 suppresses LpMYBR1‐mediated transcriptional activation of LpNRT1.5 via phosphorylation, thereby regulating NO ₃ ⁻ transport and leaf coloration in perennial ryegrass. These findings provide insights and offer candidate genes for turfgrass quality improvement.

Drought is one of the key factors limiting the yield and quality of Lolium perenne L. NAC transcription factors are a plant-specific family that is widely involved in plant responses to abiotic stresses such as drought. In this study, we analyzed the subcellular localization, transcriptional activity, and phenotype of LpNAC11-overexpressing plants to elucidate its role in the drought stress response of perennial ryegrass. The results showed that the LpNAC11 protein localizes to the nucleus and has transcriptional activation activity. Under drought conditions, LpNAC11-overexpressing plants exhibited better growth and higher survival rates than WT plants. In these overexpressing plants, leaf relative water content and proline content were significantly increased, whereas electrical conductivity and malondialdehyde (MDA) content were significantly decreased. Furthermore, histochemical staining revealed that LpNAC11-overexpressing plants accumulated fewer reactive oxygen species under drought treatment. Quantitative real-time PCR (qPCR) analysis revealed that the expression levels of multiple drought stress-responsive genes were significantly upregulated in the LpNAC11-overexpressing plants. This study reveals the function of LpNAC11 in regulating the drought stress response in perennial ryegrass, providing a theoretical foundation for breeding drought-tolerant cultivars in this species.

The improvement of plant architecture has historically driven the “Green Revolution,” leading to substantial increases in cereal yields. However, the architectural requirements for turfgrass differ from those of cereals. Despite its importance, our understanding of the mechanisms that regulate plant architecture in turfgrass remains limited. In this study, we identified that Lolium perenne BTB/POZ-MATH5.6s (LpBPM5.6) plays a crucial role in shaping the ideal turfgrass architecture by influencing the stability of ALTERED RESPONSE TO GRAVITY 1 (LpARG1) in perennial ryegrass. The phenotypes of LpBPM5.6-overexpressing and RNA interference transgenic seedlings revealed that knocking down LpBPM5.6 results in an ideal turfgrass architecture characterized by reduced plant height, increased tillering, and fewer fibrous roots. Through yeast 2-hybrid library screening, as well as subsequent pull-down, luciferase complementation imaging, and co-immunoprecipitation techniques, we demonstrated that LpBPM5.6 interacts with LpARG1. Protein degradation assays indicated that LpBPM5.6 regulates the degradation of LpARG1. Additionally, microscopic observations showed that LpBPM5.6 and LpARG1 affect the trafficking of PIN1, thereby influencing auxin transport and turfgrass architecture. This study elucidates how LpBPM5.6 regulates turfgrass architecture by modulating the stability of LpARG1, providing insights into the genetic mechanisms underlying the ideal plant architecture for turfgrass.

Drought serves as a major environmental stress that restricts both the yield and quality of perennial ryegrass. Therefore, it is important to identify the essential genes that determine drought tolerance in perennial ryegrasses. In this paper, we isolated a drought‐induced NAC transcription factor LpNAC22. Transcriptional activity assays in yeast and plant cells indicated that LpNAC22 has transcriptional activation function. Subcellular localization observations revealed that LpNAC22 localized in the nucleus, compatible with its function as a transcription factor. LpNAC22 overexpression plants had enhanced drought tolerance and reduced cell membrane damage, whereas the knockdown of LpNAC22 in perennial ryegrass reduced plant drought tolerance and led to aggravated cell membrane damage. Late embryogenesis abundant (LEA), well‐known stress resistance proteins, can protect the cell membrane from damage during drought conditions. DNA affinity purification sequencing and transcriptional regulation analysis demonstrated that LpNAC22 upregulates two LEA family genes, LpLEA1 and LpLEA2‐1, by directly binding to their promoters. Furthermore, we demonstrated that overexpression of LpLEA1 and LpLEA2‐1 in Arabidopsis enhanced drought tolerance and reduced cell membrane damage under drought conditions. Our findings provide evidence that LpNAC22 improves drought resistance by modulating the transcription of LEA family genes in perennial ryegrass.

Microtubules play pivotal roles in establishing trichome branching patterns, which is a model system for studying cell-shape control in Arabidopsis (Arabidopsis thaliana). However, the signaling pathway that regulates microtubule reorganization during trichome branching remains poorly understood. In this study, we report that MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) is involved in GLABRA3 (GL3)-mediated trichome branching by regulating microtubule stability. Loss of MDP25 function led to excessive trichome branching, and this phenotype in mdp25 could not be rescued by the MDP25 K7A or MDP25 K18A mutated variants. Pharmacological treatment and live-cell imaging revealed increased microtubule stability in the mdp25 mutant. Furthermore, the microtubule collar observed during trichome branching remained more intact in mdp25 compared to the WT under oryzalin treatment. Results of genetic assays further demonstrated that knocking out MDP25 rescued the reduced branching phenotype of gl3 trichomes. In gl3 trichomes, normal microtubule organization was disrupted, and microtubule stability was significantly compromised. Moreover, GL3 physically bound to the MDP25 promoter, thereby inhibiting its expression. Overexpression of GL3 negated the effects of P_MDP25-driven MDP25 or its mutant proteins on trichome branching and microtubules in the mdp25 background. Overall, our study uncovers a mechanism by which GL3 inhibits MDP25 transcription, thereby influencing microtubule stability and regulating trichome branching. This mechanism provides a connection between early regulatory components and microtubules during trichome development.

Background: Drought resistance is a complex characteristic closely related to the severity and duration of stress. Perennial ryegrass (Lolium perenne L.) has no distinct drought tolerance but often encounters drought stress seasonally. Although the response of perennial ryegrass to either extreme or moderate drought stress has been investigated, a comprehensive understanding of perennial ryegrass response to both conditions of drought stress is currently lacking. Results: In this study, we investigated the genetic variation in drought resistance in 18 perennial ryegrass varieties under both extreme and moderate drought conditions. The performance of these varieties exhibited obvious diversity, and the survival of perennial ryegrass under severe stress was not equal to good growth under moderate drought stress. ‘Sopin’, with superior performance under both stress conditions, was the best-performing variety. Transcriptome, physiological, and molecular analyses revealed that ‘Sopin’ adapted to drought stress through multiple sophisticated mechanisms. Under stress conditions, starch and sugar metabolic enzymes were highly expressed, while CslA was expressed at low levels in ‘Sopin’, promoting starch degradation and soluble sugar accumulation. The expression and activity of superoxide dismutase were significantly higher in ‘Sopin’, while the activity of peroxidase was lower, allowing for ‘Sopin’ to maintain a better balance between maintaining ROS signal transduction and alleviating oxidative damage. Furthermore, drought stress-related transcriptional and posttranscriptional regulatory mechanisms, including the upregulation of transcription factors, kinases, and E3 ubiquitin ligases, facilitate abscisic acid and stress signal transduction. Conclusion: Our study provides insights into the resistance of perennial ryegrass to both extreme and moderate droughts and the underlying mechanisms by which perennial ryegrass adapts to drought conditions.