Subham Bhakta, Himanshu Tak, Sanjana Negi, Vitthal Barvkar, Namrata Waghamare, Anand Ballal, Sudhir Singh
Stress induced senescence (SIS)-associated yield loss is a major setback in banana cultivation. Despite being grown all over the world, a detailed understanding of the senescence-related transcription factors is hitherto limited in this vital cash crop. NAC (NAM, ATAF and CUC) family proteins are postulated to be the major players that regulate senescence-pathways in model plants. Herein, we have identified and characterised a master regulator of SIS, MaNAC46, which integrates stress-signals into jasmonic acid-dependent senescence-pathway in Musa x paradisiaca cv. Rasthali (genome composition, AAB). BLAST-analysis showed NAC46 gene to be located on chromosome-6 of the Musa acuminata genome. As this NAC clustered tightly with AtNAC46 in the phylogenetic-analysis, it was annotated as MaNAC46 (Figure S1B). This protein displayed the presence of a conserved N-terminal NAC domain and a C-terminal variable-domain (Figure S1A). Expression-profiling detected strong MaNAC46 transcripts at 3H and 12H post treatment with 250 mM NaCl. On long-term exposure, the NAC46 transcript peaked around 5 or 10 days for salinity or drought stress, respectively. Subsequently, its expression largely declined over time with some fluctuations observed around the 20–25th day [Figure 1A(i),(ii) and Figure S2A–D]. A similar pattern was reported for the consortium of senescence-NACs (SenNACs) transcripts, which too showed positive-to-negative inversions in expression with time. These fluctuations in SenNACs have been shown to be necessary for averting untimely senescence during stress (Kim et al. 2018). To visualise its tissue-specific induction, the 5′-regulatory region of the MaNAC46 was ligated upstream of the GUS-reporter gene and transformed into tobacco leaf-disks to generate ProMaNAC46-GUS tobacco lines (Figure S3). Under ambient conditions, the ProMaNAC46-GUS activity was primarily detected in the vascular regions. However, exposure to abiotic stresses, which are vital for the onset of senescence, led to the spread of the promoter-driven GUS activity in various tissues. Strong GUS activity was detected after 1H drought, 1H salinity, or 24H cold [Figure 1A(iii), Figure S3C]. Transgenic banana plants (Musa x paradisiaca cv Rasthali, developed from the embryogenic cell-suspension derived from male flower-bud) constitutively-expressing MaNAC46 driven by ProZmUbi displayed severe growth-retardation, premature leaf-yellowing/wilting, and early senescence [Figure 1B(i), Figure S4]. qRT-PCR demonstrated 50–500 folds enhanced expression of MaNAC46-transcripts in three distinct MaNAC46 overexpressing (Ox)-lines (L1, L2, L3) [Figure 1B(ii)]. CRISPR-Cas9 mediated MaNAC46-knockout (KO) lines were generated by targeting the C-terminal activation-domain with a specific gRNA employing the pRGEB31-vector (Figure S5A). Indels at target-loci were identified in three lines, that is, a single T-insertion 3 bp upstream of the PAM-site leading to frame disruption in KO-L1, while omission of a single-codon was observed in the remaining two lines (Figure S5B–D). In contrast to the control plants, the MaNAC46-KO L1 plants showed delayed natural-senescence and even retained most of their early emerged leaves [Figure 1B(iii),(iv)]. Chlorophyll-catabolism, a hallmark of leaf-senescence, is regulated by NAC-transcription factors such as AtNAP, ORE1, and AtNAC016 in model plants (Negi et al. 2023). MaNAC46-Ox banana leaves exhibited extreme yellowing and wilting, with significantly reduced total chlorophyll content, whereas KO-lines retained greener leaves and ~2-fold higher chlorophyll levels [Figure 1B(v), Figure S6A,B]. These observations resonated very well with the delayed senescent phenotype observed in the KO-lines. In line with the enhanced chlorophyll content, two key chlorophyll-catabolic genes, viz. NYC and PPH, were markedly upregulated in Ox-lines but downregulated in KO-plants [Figure 1B(vi)]. A reduction in NYC/PPH expression and a noticeable increase in total chlorophyll content also correlated with the enhanced photosynthetic efficiency (Fv/Fm) observed in the leaves of KO-plants (Figure S6C). Taken together, this data strongly indicates that MaNAC46 regulates chlorophyll-catabolism. Autophagy facilitates nutrient-recycling during plant-senescence. Transmission electron-microscopy revealed abundant autophagic body-like double-membrane structures in MaNAC46-Ox leaves, which were normally absent in control [Figure 1C(i)]. Transcriptional-regulation of autophagy is poorly understood; the available information is largely focused on the role of Autophagy-Related-Genes (ATGs), which in turn are controlled by PIF4 and bHLH4 (Lee et al. 2025). Among crop plants, ATG2 is proposed to play a role in autophagy-senescence cross-talk in soybean (Liao et al. 2022). Interestingly, in banana, MaNAC46-Ox lines showed strong induction of ATG4, ATG5, ATG6, ATG8B, ATG8E, and ATG12, whereas these genes were repressed in KO-lines [Figure 1C(ii)]. These observations clearly demonstrate that MaNAC46 regulates autophagy-related genes during senescence. This is the first time a NAC-transcription factor has been implicated in regulating autophagy-dependent senescence in banana and, more broadly, in plants. TEM-analysis also demonstrated a substantial number of plastoglobulins in the chloroplast of Ox-line [Figure 1C(i)], which was in unison with the precocious senescence and retarded growth phenotype observed in these lines. Enhanced levels of ROS are known to exacerbate the generation of plastoglobulins during senescence. Intense H2-DCFDA staining of Ox-lines and substantially higher content of H2O2, indicative of a ROS burst, lend credence to the notion that the biogenesis of plastoglobulins in chloroplasts was indeed due to NAC46-induced senescence [Figure 1D(i), Figure S7]. Reduced expression of catalase (CAT), peroxiredoxin (PRXDN), and thioredoxin-peroxidases (TDP) in Ox-lines, and the higher transcript abundance of these genes in the KO-line, also points to the role of NAC46 in modulating ROS burst/levels in banana [Figure 1D(ii)]. Abscisic-acid (ABA), jasmonic-acid (JA) and salicylic-acid (SA) are the major stress-hormones linked with induction of senescence (Kim et al. 2018). In MaNAC46-KO banana plants, levels of JA-isoleucine (a bioactive JA-derivative) were significantly reduced, while ABA and SA levels remained comparable to those in control plants [Figure 1E(i), Figure S8A], indicating a specific role for MaNAC46 in JA-mediated senescence. Supporting this, the JA-biosynthetic genes 13S-LOX and Allene oxide-cyclase (AOC) were repressed in KO-lines but strongly induced in MaNAC46-Ox lines [Figure 1E(ii), Figure S8B]. Finally, to verify the involvement of hormones in activation of MaNAC46, ProMaNAC46 harbouring tobacco-plants were exposed to ABA, SA, Methyl-jasmonate and ethephon. Pro-MaNAC46 driven GUS-expression was distinctly visualised after 12H of ABA/SA or 3H of MeJA treatment [Figure 1E(iii), Figure S8C]. Interestingly, the strongest change in ProMaNAC46-GUS was observed upon treatment with MeJA; whereas, in contrast, ethephon suppressed GUS-activity in these lines. Collectively, MaNAC46 was shown to coordinate multiple stress-signals into jasmonic acid-dependent senescence and bring about SIS by integrating autophagy (ATGs), chlorophyll catabolism (NYC/PPH), ROS burst and jasmonic acid biosynthesis (LOX/AOC) (Figure S9). This work has tremendous biotechnological implications as MaNAC46 can be potentially targeted to evade SIS, and consequently improve the yield. It is reported that the delayed senescence-phenotype in starchy fruit crops increases yield (Zhang et al. 2021). It will be of great interest to analyse the MaNAC46-KO lines for their potential to resist SIS-mediated yield reduction in banana. These findings open new avenues for both basic as well as applied aspects of banana crop improvement through the modulation of senescence. S.B., H.T., A.B., S.S. designed experiments. S.B., H.T., S.N., V.B., N.W. conducted experiments. S.B., H.T., A.B., S.S. analysed data and wrote manuscript. The work was supported by funding from the Department of Atomic Energy, Government of India. This work was supported by the Department of Atomic Energy, Government of India. The authors declare no conflicts of interest. Data underlying this article are available in the article and its online Supporting Information. Figures S1–S9: pbi70552-sup-0001-FigureS1-S9.pptx. Table S1: Primers for cloning and expression work. Table-S2. Primers for expression-analysis. Table-S3. Sequence of MaNAC46 and ProMaNAC46. Data S1: Materials and Methods. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.