PlantView Supports Plant Senescence Research: The Module WmABF1-1-WmMYB11l Manipulates the LeafSenescence of Welwitschia by Integrative Regulation ofAbscisic Acid Biosynthesis, Chlorophyll Degradatio

New progress has been made in understanding the ABA-independent regulation of leaf senescence mediated by WmMYB111.

Researchers Tao Wan and Qingfeng Wang from the Wuhan Botanical Garden, Chinese Academy of Sciences, have made new progress in elucidating the regulatory mechanisms of leaf senescence in Welwitschia mirabilis. Their findings were published in “Plant Biotechnology Journal”  (IF = 10.5, Q1 top journal).

This study reveals a WmABF1-1–WmMYB111 regulatory module that integrates ABA accumulation, chlorophyll degradation, and nitrogen transport to regulate leaf senescence in W. mirabilis, providing new insights into the molecular mechanisms underlying its unique leaf senescence pattern.

Welwitschia mirabilis is an ancient gymnosperm famous for its two leaves, which can survive for more than a thousand years, and its ability to adapt to the extremely arid environment of the Namib Desert. Although considerable research has focused on the drought resistance mechanisms of its leaves, little is known about the underlying regulatory mechanisms of leaf senescence. Unlike the typical pattern in which plant leaves senesce from the tip toward the base, senescence in W. mirabilis is restricted to the leaf tip (S6 region), while the remaining regions remain green, suggesting a unique programmed senescence pathway. Physiological analyses showed that the senescent region (S6) exhibited significantly increased abscisic acid (ABA) levels, increased electrolyte leakage, and decreased chlorophyll content. At the molecular level, the expression of key ABA biosynthesis genes (WmNCED6, WmNCED9), senescence-associated genes (WmSEN1, WmSEN2), and chlorophyll degradation genes (WmPAO1-1, WmPAO2-1) was upregulated in the S6 region, indicating that ABA accumulation is a core factor driving senescence.

Previous studies have proposed that continuous leaf growth is regulated by the KNOX-PHAN gene module, but the transcriptional regulatory network underlying senescence remains unclear. As a key hormone regulating leaf senescence, ABA biosynthesis and signaling pathways have not been extensively explored in W. mirabilis. By screening senescence-induced promoter cis-elements, the researchers found that MYB-binding sites (MBSs) were highly enriched in the promoters of ABA biosynthesis genes (WmNCEDs), leading to the identification of the R2R3-MYB transcription factor WmMYB111. Its expression was induced by ABA and synchronized with the progression of senescence, suggesting a central regulatory role. Further analyses showed that WmABF1-1, a key component of the ABA signaling pathway and a member of the ABF family, directly activates WmMYB111, forming a WmABF1-1–WmMYB111 regulatory module. This module amplifies ABA signaling in a cascade and integrates chlorophyll degradation with nitrogen (N) transport processes (Figure 1).

In addition, leaf senescence is accompanied by nitrogen redistribution. The nitrogen content of the senescent region was significantly reduced, while nitrogen transport genes (WmNRT1.7a, WmNRT2.5) were activated. Low-nitrogen conditions accelerated senescence, whereas high-nitrogen conditions delayed it, confirming the close relationship between senescence and nutrient recycling. WmABF1-1 and WmMYB111 could directly activate the promoters of WmNRTs, indicating that this module coordinates ABA accumulation, chlorophyll degradation, and nitrogen transport to optimize resource utilization during senescence.

To verify whether the W. mirabilis ABA biosynthesis genes WmNCED6 and WmNCED9 can drive senescence, the researchers overexpressed these two genes in Arabidopsis thaliana (Col-0 wild-type background), generating the transgenic lines WmNCED6-OE#1,2 and WmNCED9-OE#1,2. The senescence phenotypes of the third and fourth rosette leaves were examined, and photosynthetic efficiency (Fv/Fm) was measured using the PlantView230F modulated chlorophyll fluorescence in vivo imaging system from Guangzhou Biolight Biotechnology Co., Ltd. (Figure 2). Compared with the wild type, all overexpression lines exhibited significantly accelerated leaf senescence, characterized by extensive leaf yellowing and wilting. Meanwhile, their Fv/Fm ratios, a key indicator of photosynthetic performance, decreased sharply, with the average value declining from 0.78 ± 0.02 in the wild type to 0.53 ± 0.03 (p < 0.01), indicating severe impairment of photosynthetic function caused by chlorophyll degradation.

Similarly, to investigate the regulatory role of the W. mirabilis transcription factor WmMYB111 in senescence, the researchers analyzed the senescence phenotypes and Fv/Fm ratios of the third and fourth rosette leaves of Arabidopsis wild-type plants (Col-0) and WmMYB111-overexpressing lines (WmMYB111-OE#1,2) (Figure 3). The results showed that overexpression of WmMYB111 significantly delayed leaf senescence. Compared with the obvious yellowing and wilting observed in wild-type leaves, the leaves of the transgenic lines remained green for a longer period, and their Fv/Fm ratios, a key indicator of photosynthetic function, were significantly higher than those of the control group, based on multiple biological replicates.

To investigate the function of WmABF1-1 in leaf senescence of W. mirabilis, TRV-mediated gene silencing was used to generate WmABF1-1 knockdown lines (TRV-WmABF1-1) and an empty-vector control (TRV-control). The senescence progression of detached leaf discs was monitored over a 20-day period (Figure 4). The results showed that silencing WmABF1-1 significantly delayed senescence. By day 15, the TRV-control leaf discs showed obvious yellowing and wilting, with more than 80% of the area becoming yellow, whereas the TRV-WmABF1-1 leaf discs still maintained more than 60% green area by day 20. Concurrent measurements of chlorophyll fluorescence efficiency (Fv/Fm) further confirmed that the Fv/Fm value of the TRV-control group continuously decreased from an initial 0.82 ± 0.03 to 0.35 ± 0.05 on day 20, representing a 57.3% reduction, while the silenced group decreased only to 0.68 ± 0.04 over the same period, representing a 17.1% reduction (P < 0.001). These results directly demonstrate that WmABF1-1 positively regulates senescence by promoting chlorophyll degradation and cellular disassembly programs.

In senescent leaf segments of the ABA-deficient mutant aba1-5, the researchers transiently expressed the ABA signaling factor WmABF1-1 and the senescence-suppressing factor WmMYB111 to analyze local phenotypes and photosynthetic efficiency (Fv/Fm) on day 10 (Figure 4). The results showed that WmMYB111 overexpression significantly delayed senescence. The treated leaf regions exhibited substantially less yellowing, and their Fv/Fm values were significantly higher than those of the control group, reaching levels comparable to the basal region, directly demonstrating that WmMYB111 suppresses the execution of the senescence program. In contrast, WmABF1-1 overexpression accelerated senescence, resulting in decreased Fv/Fm values and more severe leaf wilting.

Experiments using PlantView

Furthermore, to determine the ability of the WmMYB111 transcription factor to activate its target gene promoters, clarify the regulatory function of WmABF1-1 on core senescence-related genes in W. mirabilis, and investigate the regulatory mechanisms of WmABF1-1 and WmMYB111 on the nitrogen transport genes WmNRT1.7a and WmNRT2.5, dual-luciferase assays were performed in Nicotiana benthamiana leaves (Figure 5). Using the Chlorophyll Fluorescence In Vivo Imaging System PlantView230F from Guangzhou Biolight Biotechnology, the researchers found that WmMYB111 significantly enhanced the activity of all target gene promoters. WmABF1-1 also significantly activated all three types of promoters. WmMYB111 significantly activated the promoter activities of all nitrogen transport genes, whereas WmABF1-1 strongly suppressed the activities of these nitrogen transport gene promoters.

Taken together, these results demonstrate that overexpression of WmNCED6/9 can directly trigger premature senescence in a model plant by promoting ABA biosynthesis. This not only confirms their central roles in leaf-tip senescence in W. mirabilis, but also provides cross-species functional evidence for the “ABA accumulation → senescence execution” pathway.

As a negative regulator of senescence, WmMYB111 effectively suppresses chlorophyll degradation and maintains the integrity of the photosynthetic apparatus, thereby delaying the senescence process. The findings also reveal WmMYB111 as a key downstream negative regulator of senescence in the ABA pathway, capable of maintaining photosynthetic function and nutrient retention through an ABA-independent mechanism.

DOI: 10.1111/pbi.70290