Expression of chlorophyll (Chl) catabolic genes during leaf senescence is tightly controlled at the transcriptional level. Here, we identified a NAC family transcription factor, LpNAL, involved in regulating Chl catabolic genes via the yeast one-hybrid system based on truncated promoter analysis of STAYGREEN (LpSGR) in perennial ryegrass (Lolium perenne L.). LpNAL was found to be a transcriptional repressor, directly repressing LpSGR as well as the Chl b reductase gene, NONYELLOWING COLORING1. Perennial ryegrass plants over-expressing LpNAL exhibited delayed leaf senescence or stay-green phenotypes, whereas knocking down LpNAL using RNA interference accelerated leaf senescence. Comparative transcriptome analysis of leaves at 30 d after emergence in wild-type, LpNAL-overexpression, and knock-down transgenic plants revealed that LpNAL-regulated stay-green phenotypes possess altered light reactions of photosynthesis, antioxidant metabolism, ABA and ethylene synthesis and signaling, and Chl catabolism. Collectively, the transcriptional repressor LpNAL targets both Chl a and Chl b catabolic genes and acts as a brake to fine-tune the rate of Chl degradation during leaf senescence in perennial ryegrass.

Fine tuning the progression of leaf senescence is important for plant fitness in nature, while the “staygreen” phenotype with delayed leaf senescence has been considered a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass (Panicum virgatum L.) CCCH-type Zinc finger gene, Strong Staygreen (PvSSG), was characterized as a suppressor of leaf senescence as overexpression or suppression of the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll (Chl) catabolic pathway genes were involved in the PvSSG-regulated leaf senescence. PvSSG was identified as a nucleus-localized protein with no transcriptional activity. By yeast two-hybrid screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1/2 (NAC-LIKE, ACTIVATED BY AP3/PI). Overexpression of PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating Chl catabolic genes to promote Chl degradation. PvSSG, through protein–protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioning as a “brake” in the progression of leaf senescence. Moreover, overexpression of PvSSG resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops.

Lolium multiflorum is one of the world-famous forage grasses with rich biomass, fast growth rate and good nutritional quality. However, its growth and forage yield are often affected by drought, which is a major natural disaster all over the world. MYB transcription factors have some specific roles in response to drought stress, such as regulation of stomatal development and density, control of cell wall and root development. However, the biological function of MYB in L. multiflorum remains unclear. Previously, we elucidated the role of LmMYB1 in enhancing osmotic stress resistance in Saccharomyces cerevisiae. Here, this study elucidates the biological function of LmMYB1 in enhancing plant drought tolerance through an ABA-dependent pathway involving the regulation of cell wall development and stomatal density. After drought stress and ABA stress, the expression of LmMYB1 in L. multiflorum was significantly increased. Overexpression of LmMYB1 increased the survival rate of Arabidopsis thaliana under drought stress. Under drought conditions, expression levels of drought-responsive genes such as AtRD22, AtRAB and AtAREB were up-regulated in OE compared with those in WT. Further observation showed that the stomatal density of OE was reduced, which was associated with the up-regulated expression of cell wall-related pathway genes in the RNA-Seq results. In conclusion, this study confirmed the biological function of LmMYB1 in improving drought tolerance by mediating cell wall development through the ABA-dependent pathway and thereby affecting stomatal density.

Finetuning the progression of leaf senescence is important for plant’s fitness in nature, while ‘staygreen’ with delayed leaf senescence has been considered as a valuable agronomic trait in crop genetic improvement. In this study, a switchgrass CCCH-type Zinc finger gene, PvC3H29 , was characterized as a suppressor of leaf senescence that over-expressing or suppressing the gene led to delayed or accelerated leaf senescence, respectively. Transcriptomic analysis marked that chlorophyll catabolic pathway genes were involved in the PvC3H29-regulated leaf senescence. PvC3H29 was a nucleus-localized protein with no transcriptional activity. By Y2H screening, we identified its interacting proteins, including a pair of paralogous transcription factors, PvNAP1&2. Over-expressing the PvNAPs led to precocious leaf senescence at least partially by directly targeting and transactivating chlorophyll catabolic genes to promote chlorophyll degradation. PvC3H29, through protein-protein interaction, repressed the DNA-binding efficiency of PvNAPs and alleviated its transactivating effect on downstream genes, thereby functioned as a ‘brake’ in the progression of leaf senescence. Moreover, over-expressing PvC3H29 resulted in up to 47% higher biomass yield and improved biomass feedstock quality, reiterating the importance of leaf senescence regulation in the genetic improvement of switchgrass and other feedstock crops. One-sentence summary: PvC3H29 interacts with transcription factors, PvNAP1&2, to inhibit their transactivation on chlorophyll catabolism and leaf senescence in switchgrass.

Perennial ryegrass (Lolium perenne L.) is a vital cool-season forage and turfgrass species whose productivity is increasingly threatened by climate change-induced abiotic stresses. The Heat Shock Factor (HSF) family are key transcription factors known to mediate plant stress responses, yet their functional landscape in perennial ryegrass remains poorly characterized, especially beyond heat stress. In this study, genome-wide identification revealed 26 LpHSF genes in perennial ryegrass, harboring abundant stress-responsive cis-elements. Moving beyond a singular focus on thermotolerance, the study established a comprehensive spatio-temporal expression atlas of LpHSFs. The findings demonstrate that specific LpHSFs, particularly members of the LpHSFA2 subclass, LpHSFA3, LpHSFC1.3, and LpHSFC1.4, are significantly upregulated under a broad spectrum of abiotic stresses, including salinity, alkalinity, and heavy metal exposure, indicating their roles as broad-spectrum stress regulators. Furthermore, this study uncovered distinct tissue-specific expression patterns, with Class A genes predominantly expressed in leaves and Classes B and C in roots and crowns, implicating their roles in organ development and stress adaptation. Notably, the expression of numerous LpHSFs during leaf senescence and their induction by hormones such as ABA and MeJA further highlights their functional diversity. This study provides the first integrated expression atlas of the LpHSF family, revealing their versatile roles in coordinating responses to diverse environmental and developmental signals. This work lays a crucial foundation for future functional studies and positions LpHSFs as prime targets for molecular breeding aimed at enhancing multi-stress resilience in perennial ryegrass.