A truncated ETHYLENE INSENSITIVE3-like protein, GhLYI, regulates senescence in cotton

Abstract

Numerous endogenous and environmental signals regulate the intricate and highly orchestrated process of plant senescence. Ethylene (ET), which accumulates as senescence progresses, is a major promoter of leaf senescence. The master transcription activator ETHYLENE INSENSITIVE3 (EIN3) activates the expression of a wide range of downstream genes during leaf senescence. Here, we found that a unique EIN3-LIKE 1 (EIL1) gene, cotton LINT YIELD INCREASING (GhLYI), encodes a truncated EIN3 protein in upland cotton (Gossypium hirsutum L.) that functions as an ET signal response factor and a positive regulator of senescence. Ectopic expression or overexpression of GhLYI accelerated leaf senescence in both Arabidopsis (Arabidopsis thaliana) and cotton. Cleavage under targets and tagmentation (CUT&Tag) analyses revealed that SENESCENCE-ASSOCIATED GENE 20 (SAG20) was a target of GhLYI. Electrophoretic mobility shift assay (EMSA), yeast 1-hybrid (Y1H), and dual-luciferase transient expression assay confirmed that GhLYI directly bound the promoter of SAG20 to activate its expression. Transcriptome analysis revealed that transcript levels of a series of senescence-related genes, SAG12, NAC-LIKE, ACTIVATED by APETALA 3/PISTILLATA (NAP/ANAC029), and WRKY53, are substantially induced in GhLYI overexpression plants compared with wild-type (WT) plants. Virus-induced gene silencing (VIGS) preliminarily confirmed that knockdown of GhSAG20 delayed leaf senescence. Collectively, our findings provide a regulatory module involving GhLYI-GhSAG20 in controlling senescence in cotton.

Introduction

Leaf senescence is the final stage of plant leaf growth and development and can be induced by a variety of internal factors, such as aging and phytohormones, and also external environmental factors including darkness, UV-B, nutrient limitation, salinity, drought, and high ambient temperature (Lim et al. 2007). The senescence process is initiated by chloroplast degeneration, which is followed by the catabolism of macromolecules such as nuclear acids, proteins, and lipids and finally by the degeneration of mitochondria and nuclei (Buchanan-Wollaston et al. 2005).

Ethylene is an important phytohormone that is essential for many physiological and developmental processes, such as apical hook formation, shoot and root growth, senescence, stress response, and fruit ripening (Bleecker and Kende 2000; Liu et al. 2015; Binder 2020). A series of ethylene-response mutants in Arabidopsis (Arabidopsis thaliana) were isolated by “triple response,” which consists of shortened, thickened hypocotyls and a pronounced apical hook (Bleecker et al. 1988; Guzman and Ecker 1990; Kieber 1997). Ethylene signaling is initiated upon the perception of ethylene by receptors in the endoplasm reticulum, which interact with a Raf-like protein kinase, CONSTITUTIVE TRIPLE RESPONSE 1 (CTR1), to form a negative regulator complex (Kieber et al. 1993; Ju et al. 2012). This complex inhibits the essential positive regulator of ethylene signaling, ETHYLENE INSENSITIVE2 (EIN2) (Alonso et al. 1999; Li et al. 2015). Once ethylene signaling is activated, EIN2 activates genes encoding 2 master transcription factors, ETHYLENE INSENSITIVE3 (EIN3) and its homology EIN3-LIKE 1 (EIL1), which in turn regulate multiple ethylene responses (Guo and Ecker 2003; Dolgikh et al. 2019), most often in the form of a homodimer (Solano et al. 1998). In 1997, Chao et al. identified the EIN3 gene and other EIN3-LIKE genes (EIL1, EIL2, and EIL3) in Arabidopsis (Chao et al. 1997). Over the next 25 years, EIN3/EILs have been identified in many plant species, such as tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum L.), and rice (Oryza sativa L.) (Kosugi and Ohashi 2000; Tieman et al. 2001; Mao et al. 2006). EIN3/EIL proteins are almost always located in the nucleus and have highly conserved N-terminal amino acid (aa) sequences. Some important structural forms have been identified in the first half of EIN3/EIL protein sequences, such as highly acidic N-terminus aa regions, 5 basic aa groups, and proline-rich regions (Kosugi and Ohashi 2000). Meanwhile, C-terminal sequences are less conserved than N-terminal sequences in this family. For example, the poly-asparagine or poly-glutamine regions present in the C-terminal sequences of Arabidopsis are absent in N. benthamiana (Rieu et al. 2003). Therefore, analyzing the C-terminal sequence motifs of these factors may help us understand their evolution and function differentiation. The importance of EIN3/EIL1 transcription factors in the ethylene-response pathway has been demonstrated in Arabidopsis through the elimination or severe reduction of ethylene-induced long-term growth inhibition in ein3eil1 double mutant seedlings and the constitutive ethylene-response phenotypes that result from high-level expression of EIN3 or EIL1 in Col-0 or ein2 plants (Binder et al. 2004).

In the absence of ethylene, EIN3/EIL family proteins are degraded via the 26S proteasome by EIN3 BINDING F-BOX1 (EBF1) and EIN3 BINDING F-BOX2 (EBF2) (Guo and Ecker 2003; Potuschak et al. 2003; Binder et al. 2007). Meanwhile, EBF2 transcript levels are directly induced by EIN3, forming a negative feedback regulatory loop between EIN3 and the EBFs (Konishi and Yanagisawa 2008). Beyond EBF2, multiple studies have proved that EIN3 transcriptionally activates the expression of ETHYLENE RESPONSE FACTOR (ERF1), SALICYLIC ACID INDUCTION DEFICIENT2 (SID2), HOOKLESS1 (HLS1), and ETHYLENE AND SALT-INDUCIBLE ERF GENES (ESE1), some of which have the core sequence 5′-ATGTA-3′ within their promoters (Chen et al. 2009; Zhang et al. 2011, 2018; Mao et al. 2016). Much is now known about EIN3/EIL1, however, only very limited information is available concerning the truncated versions of these proteins.

Exogenous application of ethylene accelerates leaf senescence, while application of inhibitors of ethylene biosynthesis or action delays senescence (Abeles et al. 1988). Forward genetic assays revealed EIN3 as a positive regulator of leaf senescence, with the level and activity of EIN3 mRNA being increased during the senescence process (Yu et al. 2021b). Moreover, constitutive overexpression or temporary activation of EIN3 is sufficient to accelerate leaf senescence symptoms (Wang et al. 2021a). Conversely, loss of function in Arabidopsis EIN3 and EIL1 leads to a delay in age-dependent and ethylene-, jasmonic acid (JA)-, or dark-induced leaf senescence. EIN3 transcription is also upregulated by dark treatment, a condition used to quickly induce nonnatural leaf senescence. In particular, EIN3, ORE1, and chlorophyll catabolic genes (CCGs, namely, NON-YELLOWING1 [NYE1], NON-YELLOW COLORING 1 [NYC1], and PHEOPHORBIDE A OXYGENASE [PAO]) have been shown in Arabidopsis to constitute a coherent feed-forward loop that is involved in the robust regulation of ethylene-mediated chlorophyll degradation during leaf senescence (Qiu et al. 2015). EIN3 has also been shown to repress microRNA164 (miR164) transcription by directly binding to its promoter region, leading to increased transcript levels of ORE1 which consequently promote leaf senescence (Li et al. 2013). EIN3 and EIL1 are also required for salicylic acid (SA)-induced leaf senescence in Arabidopsis. NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1), the master regulator of SA signaling, interacts with EIN3 to promote its transcriptional activity (Yu et al. 2021a).

Genes whose expression levels change during leaf senescence are referred to as leaf senescence-associated genes (SAGs) and are more specifically divided according to the direction of expression change into senescence downregulated genes and senescence upregulated genes. The latter group includes genes encoding enzymes related to chlorophyll degradation, proteases, nucleases, and lipases (Buchanan-Wollaston et al. 2005; Li et al. 2012). In 1998, researchers found that the expression of AtSAG20 gradually increased with age-dependent leaf senescence and moreover that its expression was induced under dark and ethylene treatments (Weaver et al. 1998). In addition, it has been shown that upon transfection of Arabidopsis with the extracellular fungal protein Nep1, the mRNA level of AtSAG20 increases rapidly within 15 min (Keates et al. 2003).

In 2017, our team identified 1 genomic locus (D08:3040023) in upland cotton (Gossypium hirsutum) that exhibited pleiotropic associations with lint percentage, bolls per plant, and seed size by integrating GWAS analysis with expression profiling data and gene-based association and function annotation of putative orthologs in Arabidopsis. A truncated EIL gene (Gh_D08G0312) was identified as the candidate gene responsible for this LINT YIELD INCREASING (GhLYI) and therefore named GhLYI. A nonsynonymous SNP (A/C) occurring at the 184-bp position of that gene resulted in a change from Asn to histidine (His) and was determined to be associated with the GWAS signal (Fang et al. 2017). In this study, we found that GhLYI also functions as a positive regulator of senescence progress, with its overexpression evidently hastening leaf senescence by directly upregulating GhSAG20. These findings deepen our understanding of the molecular mechanisms through which ethylene signaling regulates senescence progression.

Results

A unique truncated EIL identified in cotton

The EIN3/EIL proteins, previously identified in many other plant species, are homologous to 1 another in their primary sequences of ∼600 aa residues, especially in the N-terminal half. The N-terminus of EIN3 contains 5 small clusters of basic aa (binding domain [BD] I: 53 to 66, BD II: 88 to 94, BD III: 238 to 248, BD IV: 265 to 274, and BD V: 378 to 384), each of which has 5 to 8 Lys or Arg residues(Chao et al. 1997). We isolated this GhLYI genetic locus and determined from sequence analysis that the coding sequence (CDS) of the Gh_D08G0312 gene is 360 bp in length and encodes 120 aa, much shorter than the Arabidopsis EIN3 gene, which encodes 628 aa; moreover, it contains only the BD I (Fig. 1A).

Figure 1.

GhLYI is a unique truncated EIL protein. A) Comparison of the aa sequences of Arabidopsis EIN3 and G. hirsutum GhLYI. B) Phylogenetic analysis of EIN3/EIL family genes in Arabidopsis and upland cotton (TM-1). The numbers on the branches represent the reliability percent of Bootstrap values based on 1,000 replications. C) Transcriptome expression data for EIN3/EIL family members in TM-1.

In order to investigate the evolution of EIN3/EIL gene family members in cotton, a local BLAST comparison was conducted in COTTONGEN, a genome sequencing database of upland cotton TM-1 using the Arabidopsis AtEIN3 gene as a probe, and 19 gene sequences were identified. The phylogenetic tree of the candidate genes and Arabidopsis EIN3/EIL gene families was constructed using MEGA X software. In the evolutionary tree, there are 11 genes located in the same evolutionary branch with AtEIN3 and AtEIL1. AtEIN3 and AtEIL1 genes are homologous and have functional redundancy. Therefore, it is speculated that the 8 genes (Gh_A05G0871, Gh_D05G3883, Gh_A08G1750, Gh_D08G2099, Gh_D13G2404, Gh_A13G2005, Gh_D03G1810, and Gh_A03G2063) may have been formed by replication during evolution. The other 3 genes Gh_D08G0312, Gh_D07G1670, and Gh_D12G2800 with shorter lengths may have mutated and replicated. However, there are no genes in cotton that aggregate with AtEIL2. In addition, there are 6 genes that located in the same evolutionary branch with AtEIL3. The gene in the phylogenetic tree that is closest to AtEIL3 and has the highest node expectation is the cotton EIL3 gene (Gh_A13G1864 and Gh_D13G2252). The other 2 genes are aggregated with AtEIL4 and AtEIL5 into 1 branch, namely, the cotton EIL4 and EIL5 genes (Gh_A02G1256 and Gh_D03G0396) (Fig. 1B).

To further explore the origin of this truncated EIL gene, we identified members of the EIN3/EIL family in Arabidopsis (A. thaliana), G. hirsutum L., and 2 diploid upland cotton varieties (Gossypium arboreum L. and Gossypium raimondii L.), which yielded 6, 19, 9, and 11 genes, respectively, and constructed an unrooted evolutionary tree. Three GhLYI copies were observed in G. hirsutum, but all belonged to the D subgenome provided by G. raimondii according to the phylogenetic tree (Supplemental Fig. S1).We extracted the flanking sequences of GhLYI and submitted them to the PlantRep website (http://www.plantrep.cn/) for repeated sequence search. However, no repeated sequence was found, indicating that GhLYI was not generated by transposons. We also amplified and compared the sequences of the 3′ UTR regions of GhLYI and other cotton EIN3/EIL family members and judged that GhLYI was not produced from premature transcription termination (Supplemental Fig. S2). In addition, we queried the NCBI database for family members in other species and identified 13, 9, 9, 7, 19, and 6 respective EIN3/EIL family members in rape (Brassica napus), tomato (S. lycopersicum), rice (O. sativa), poplar (Populus trichocarpa), tobacco (N. benthamiana), and grape (Vitis vinifera) (Supplemental Table S1). In these other species, the longest and shortest proteins encoded by these genes are 676 aa in S. lycopersicum and 191 aa in B. napus, respectively. The differences in length among these EIN3/EIL proteins are mainly due to deletion of the C-terminal end, which suggests that the C-terminal portion is less conserved. Ruan et al. (2018) previously identified 23 EIN3/EIL gene family members in B. napus, among which 9 lacked the BD III, BD IV, and BD V domains, indicating that such truncated EIN3/EIL proteins are commonly present in plants. However, what is less clear is the function these truncated EIN3/EIL proteins serve. In the process of species evolution, our GhLYI protein may have lost its C-terminal sequence fragment, resulting in partial functional differentiation.

To obtain information of the expression and functional differentiation of cotton EIN3/EIL gene family members, we collected the expression data of 19 members in different tissues and organs in transcriptome data from G. hirsutum L. acc. TM-1 and mapped the transcriptome expression profile (Fig. 1C) (Zhang et al. 2015a). The genes Gh_A06G0989, Gh_A02G1256, Gh_D03G0396, Gh_D12G2800, and Gh_D07G1670 are hardly expressed in root, stem, leaf, stamen, pistil, ovule, and fiber; in contrast, Gh_D13G2404, Gh_A13G2005, Gh_A08G1750, Gh_D08G2099, Gh_A05G0871, and Gh_D05G3883 are highly expressed in several tissues, especially the root and the 1 d postanthesis (dpa) ovule. Moreover, expression of Gh_D08G0312 is high in the stamen and −1 to 1 dpa ovules.

The truncated EIL is an ethylene signal response factor

Being typical plant transcription factors, many previous studies have shown that most EIN3/EIL family members localize to the nucleus (He et al. 2017). To investigate the subcellular localization of the truncated EIL, a GhLYI-GFP fusion construct was created. The signal from this fusion construct was detected not only in the nucleus but also on the membrane, whereas that from the pBinGFP4 empty vector was observed throughout the cell (Fig. 2A).

Figure 2.

The truncated EIL is an ethylene signal response factor. A) Subcellular localization of GhLYI in N. benthamiana leaves. A GhLYI-GFP fusion protein driven by the 35S promoter was transformed into the leaves. GFP protein was used as control. Aquaporin NIP2-1-RFP fusion protein was used as plasma membrane marker. H2B-RFP fusion protein was used as nuclear marker. Scale bar, 50 μm. RFP, red fluorescent protein; GhLYI-GFP, GhLYI and GFP fusion protein. B) Three-day-old etiolated seedlings overexpressing GhLYI in WT (Col-0) and ein3eil1 mutant backgrounds grown in agar plates with 5 or 10 μM ACC. Scale bar, 2 cm. LYIox/ein3eil1#1#2#3: 3 A. thaliana lines with heterologous overexpression of GhLYI in ein3eil1 mutant; JK-9 and JK-11: 2 A. thaliana lines with heterologous overexpression of GhLYI in Col-0. C) Quantification of hypocotyl lengths. Each bar represents the average length (± Sd) of at least 20 seedlings. D) The expression level of GhLYI in Arabidopsis overexpressing GhLYI in WT (Col-0) and ein3eil1 mutant backgrounds. Data are means (± Sd) of 3 biological replicates. E) Expression analysis of GhLYI after various 10 μM ACC treatment periods. Deionized water was used as control. Error bars represent standard errors of 3 independent repetitions. F) Determination of ACC content in different tissues of GhLYI-EEL transgenic and control plants. Error bars represent standard error of 3 replicates. Double asterisks show significant difference from the mock treatment at P < 0.01 (2-tailed Student's t-test) in C) to F).

To further evaluate the role of GhLYI in the ethylene signaling pathway, transgenic GhLYI was ectopically expressed under the control of the constitutive CaMV 35S promoter in Col-0 (GhLYI-EELs) and ein3 eil1 (LYIox). The ein3 eil1 double mutant displayed strong ethylene insensitivity phenotypes in terms of the triple response. Overexpression of GhLYI in the ein3 eil1 background (LYIox) could compensate for the phenotype of mutant, manifested by remarkably shorter hypocotyls and roots than ein3 eil1 upon 1-aminocyclopropane-1-carboxylic acid (ACC) treatment (Fig. 2B). Plants with the highest level of transgene expression had the most obvious ethylene triple response phenotype (Fig. 2, C and D). Compared with the WT, etiolated seedlings of GhLYI-EELs displayed a more ethylene-responsive phenotype in shoots and roots (Fig. 2B). As the concentration of ACC increased, the hypocotyl length of GhLYI-EELs further shortened (Fig. 2, C and D).

In addition, we measured ACC content in GhLYI overexpression transgenic cottons (GhLYIOXs, T2G-45) and found that the leaves, stems, and buds of transgenic plants had significantly lower contents than those of control plants (Fig. 2E). This may be due to the increase of GhLYI protein leading to an increase in sensitivity to ethylene, which in turn inhibits ethylene synthesis. Previous reports have shown that after treating WT Arabidopsis with exogenous ethylene or ACC, the mRNA level of EIN3 was not affected, but the protein content accumulated (Chao et al. 1997; Shi et al. 2003). To investigate whether GhLYI expression at transcriptional level is induced by exogenous ethylene, the expression level of GhLYI was examined in 3-wk-old TM-1 seedlings under ACC (10 µM) treatment. Within 3 h after ACC treatment, GhLYI transcripts had hardly changed but reached a maximum at 6 h and then declined to the initial level at 12 h (Fig. 2F). Similarly, the water treatment group also reached its peak at 6 h and fallen to the lowest value at 12 h. It was supposed that the consistent gene expression change trend may be affected by circadian rhythm and GhLYI was not affected by ethylene at the transcription level. These results suggested that this truncated GhLYI protein still fulfills a role in the ethylene signaling pathway.

Ectopic expression of GhLYI accelerates leaf senescence in Arabidopsis

To investigate whether GhLYI plays a role in leaf senescence as other typical EIN3 proteins do, 3 transgenic Arabidopsis lines (GhLYI-EELs) having ectopic expression of GhLYI were developed. Of them, JK-9 and JK-11 were selected for subsequent experiments due to their higher expression levels by reverse transcription quantitative PCR (RT-qPCR). As shown in Fig. 3, A and B, transgenic JK-9 and JK-11 plants displayed an early-senescence phenotype in comparison to Col-0 plants under both long-day and short-day conditions (Fig. 3, A and B), suggesting that GhLYI was a positive regulator of leaf senescence. The GhLYI-EELs also exhibited earlier leaf senescence with darkness treatment (Fig. 3C) and much higher accumulations of H₂O₂ and ${\mathrm{O}}_2^{-}$ in dark-treated leaves as determined via 3,3-diaminobenzidine (DAB) staining and nitroblue tetrazolium (NBT) (Fig. 3, D and E), suggesting that darkness promotes GhLYI-mediated H₂O₂ and ${\mathrm{O}}_2^{-}$ accumulation in leaf senescence. Thus, GhLYI is a positive regulator of aging-induced senescence in Arabidopsis leaves.

Figure 3.

Ectopic overexpression of GhLYI induced early-senescence phenotypes in Arabidopsis. A and B) The senescence phenotypes of 2-mo-old Col-0 and GhLYI-EEL plants. Early onset of leaf senescence in GhLYI-EELs was observed under both short-day A) and long-day B) conditions. Scale bar, 2 cm; JK-9 and JK-11: 2 A. thaliana lines with heterologous overexpression of GhLYI in Col-0. C) The senescence phenotypes of 2-wk-old Col-0 and GhLYI-EEL plants incubated under darkness for 7 d. Scale bar, 2 cm. D) NBT staining of the third and fourth detached rosette leaves of dark-treated Col-0 and GhLYI-EEL plants. Scale bar, 0.5 cm. E) DAB staining of the third and fourth detached rosette leaves of dark-treated Col-0 and GhLYI-EEL plants. Scale bar, 0.5 cm. F) Appearance of the third and fourth rosette leaves of untreated Col-0 and GhLYI-EEL plants over time. Scale bar, 0.5 cm. G) Chlorophyll contents of the third and fourth rosette leaves of Col-0 and GhLYI-EEL plants. The experiments are repeated 3 times and representative results are presented in the figure. The data represent the mean values of 3 replicates ± Sd. H and I) RT-qPCR analysis of GhLYI and AtSAG12 gene during ACC treatment under darkness. Data are means (± Sd) of 3 biological replicates. Single and double asterisks show significant difference from the mock treatment at P < 0.05 and P < 0.01, respectively (2-tailed Student's t-test) in G) to I).

We next examined the senescence characteristics of single leaves at different ages. Leaf yellowing occurred in the third or fourth rosette leaves of GhLYI-EELs at 35 d after emergence (DAE), whereas the leaves of Col-0 plants remained green. At 42 DAE, the third and fourth rosette leaves of transgenic plants were completely yellowed, which was not observed in Col-0 plants (Fig. 3F). Leaf yellowing is caused by chloroplast decomposition and chlorophyll loss, which are typical characteristics of leaf senescence (Breeze et al. 2011). As expected, leaf chlorophyll contents decreased more quickly and evidently in GhLYI-EELs than in Col-0 (Fig. 3G). Finally, in addition to elevated GhLYI, expression of the senescence-induced gene AtSAG12 was upregulated remarkably in JK-9 and JK-11 (Fig. 3, H and I). These data reveal that GhLYI is involved in leaf senescence in Arabidopsis.

GhLYI positively regulates senescence processes in G. hirsutum

Transcript levels of GhLYI in young, mature, early senescence, and late senescence TM-1 leaves were determined by RT-qPCR. Time-course analysis of mRNA level revealed that GhLYI transcripts gradually increased during leaf development and senescence (Fig. 4A). The positive control, GhSAG12, a widely used molecular marker of leaf senescence (Pontier et al. 1999), was specifically expressed in senescing leaves. Similarly, 2 known positive regulators of leaf senescence, NAP (Lei et al. 2020) and WRKY53 (Zentgraf et al. 2010), were assessed, and their transcript levels also increased with leaf age (Fig. 4A). The detached leaves of transgenic cottons (GhLYIOXs, T2G-2 and T2G-45) exhibited more obvious dark- and ACC-induced senescence (Fig. 4B). The transgenic plants exhibited more rapid and obvious decrease of chlorophyll contents than TM-1 at 0, 2, and 4 d after treatment (Fig. 4C). Additionally, we further assayed ${\mathrm{O}}_2^{-}$ accumulation in dark-treated leaves via NBT staining and found the overexpression lines to accumulate more ${\mathrm{O}}_2^{-}$ than the control (Fig. 4D), suggesting that darkness promotes ${\mathrm{O}}_2^{-}$ accumulation during leaf senescence in these transgenic cottons. During the same period, expression of GhLYI and GhSAG12 increased more highly in leaves of T2G-2 and T2G-45 plants than TM-1 (Fig. 4, E and F). Together, these results reveal that constitutive overexpression of GhLYI leads to accelerated senescence progression in G. hirsutum.

Figure 4.

GhLYI is a SAG whose transcription and activity are markedly induced during leaf senescence. A) RT-qPCR analysis of GhLYI, GhSAG12, GhNAP, and GhWRKY53 transcript levels in TM-1 leaves at different developmental stages. Y, young leaves; M, fully expanded mature leaves; ES, early senescent leaves; LS, late senescent leaves. The data represent the mean values of 3 repetitions and the error bar shows standard error. Scale bar, 2 cm. B) The senescence phenotypes of the first true leaves of TM-1 and GhLYI overexpression transgenic cotton plants treated with ACC under darkness. Scale bar, 2 cm. T2G-2 and T2G-45: 2 transgenic cotton lines with overexpression of GhLYI. C) Measurements of chlorophyll in detached leaves of TM-1 and GhLYI overexpression plants treated with ACC under darkness. D) NBT staining of detached leaves of TM-1 and GhLYI overexpression plants with ACC treatment under darkness. The upper row shows the leaves before dark treatment, and the lower row shows the leaves after dark treatment. Each line shows 2 representative leaves. Scale bar, 1 cm. E and F) RT-qPCR analysis of GhLYI and GhSAG12 during ACC treatment under darkness. Data are means (± Sd) of 3 biological replicates. Double asterisks show significant difference from the mock treatment at P < 0.01 (2-tailed Student's t-test) in A), C), E), and F).

To further confirm the function of GhLYI in regulating plant leaf senescence, we generated loss-of-function mutants of GhLYI using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system. The sgRNA target site was designed near the initiation codon ATG of GhLYI. Two CRISPR-Cas9 edited events were identified. LYIm-1 had a single-nucleotide “T” missing at the 18th base pair of the GhLYI ORF, leading to a frame shift. And LYIm-2 had a 6-bp missing among target site, resulting in 1 aa mutation and 2 aa deletions (Supplemental Fig. S3A). Unlike transgenic overexpression plants that exhibited growth retardation, dwarfed phenotype, and even death, there was almost no difference in growth status between the edited and the control lines (Supplemental Fig. S3B). As above, the detached leaves of LYIm-1 were used to dark and ACC treatment to induce senescence. However, NBT staining and chlorophyll content measurement results showed no difference between the edited plants and the control material (Supplemental Fig. S3, C and D). We speculate that this may be due to the genetic compensation response, as RT-qPCR results showed that the expression levels of the other 2 truncated EIN3/EIL genes Gh_D07G1670 and Gh_D12G2800 in the knockout plants have increased by about 4 and 3 times, respectively (Supplemental Fig. S3E).

Multiple biological processes are involved in GhLYI-induced senescence progression

To investigate potential mechanisms underlying GhLYI-induced senescence, RNA-seq analysis was performed using the leaves and 0 dpa ovules from transgenic overexpression lines and its corresponding control. RT-qPCR performed for 3 randomly selected upregulated genes or downregulated genes validated the RNA-seq data (Supplemental Fig. S4). Principal component analysis (PCA) and Pearson correlation analysis results showed that the samples had good repeatability and can be used for subsequent analysis (Supplemental Fig. S5). In total, we identified 15,287 and 10,037 differentially expressed genes (DEGs) in transgenic leaves and ovules respectively using a 2-fold cutoff (false discovery rate [FDR] 0.05) (Fig. 5A). The 2 tissue types shared 890 simultaneously upregulated and 1,442 downregulated genes (Fig. 5B). The red dots in 2 transcriptome volcano maps represent the genes with significantly different expression levels among all detected genes (Fig. 5, C and D). Functional assessment of the DEGs by KEGG pathway analysis revealed the upregulated DEGs to be mainly involved in plant hormone signal transduction, plant–pathogen interaction, starch and sucrose metabolism, and fatty acid degradation (Fig. 5, E and F), while downregulated DEGs were mainly involved in photosynthesis, flavonoid biosynthesis, and biosynthesis of secondary metabolites (Fig. 5, G and H). These pathway associations support a role for GhLYI in regulating senescence progression.

Figure 5.

GhLYI regulates downstream leaf senescence-related genes. A) The number of DEGs between GhLYI overexpression transgenic cottons and the control. Columns 1 to 4 represent downregulated and upregulated DEGs in leaves and ovules respectively; GhLYIOX: transgenic cotton overexpressing GhLYI. B) Venn diagrams showing the common DEGs among leaves and ovules. C and D) Volcano plots showing the distribution of DEGs in pairwise comparisons among the samples: C) leaf and D) ovule. Each dot represents a gene. Four dashed lines divide the image into six regions. Upper left and upper right represent DEGs with log2FC>2 and P<10 e-15, lower left and lower right represent DEGs with log2FC>2 and P>10 e-15, upper middle represents DEGs with log2FC<2 and P<10 e-15, and lower middle represents non-DEGs. E to H) KEGG pathway analysis of DEGs. Only the top 25 significantly enriched (P < 0.05) pathways are shown. Column color indicates significance (adjusted P-value), and the length corresponds to the proportion of DEGs with the corresponding annotation. E to H) show the KEGG pathway analysis of upregulated and downregulated DEGs in leaves and ovules, respectively.

Genome-wide identification of GhLYI-binding sites

The EIN3-binding motif sequence that was determined and tested previously is (A/T)(T/C)G(A/C/T)A(T/C/G)(C/G)T(T/G) (Konishi and Yanagisawa 2008; Zhang et al. 2011). Earlier work on the AtEIN3 protein identified 82 to 352 aa and 174 to 306 aa as the optimal and core DNA-binding domains (DBDs) respectively and also that the binding affinity of EIN3 is affected by both the number of EIN3-binding sites (EBSs) and the spacing between EBSs (Song et al. 2015). To identify the genome-wide binding sites of GhLYI, we applied cleavage under targets and tagmentation (CUT&Tag), an enzyme-tethering strategy that provides high-resolution sequencing libraries for efficient profiling of diverse chromatin components (Kaya-Okur et al. 2019). In the CUT&Tag assay using transgenic Arabidopsis, GFP antibody was used to pull down putative GhLYI-bound DNA from the leaf tissues. After the extraction of plant nuclei, DAPI staining detected the quality of the nuclei. Agarose gel electrophoresis showed that the DNA fragments of the constructed library were 200 to 700 bp (Supplemental Fig. S6). After sequencing, we successfully obtained 139, 38, and 138 binding peaks respectively in 3 biological replicates (Supplemental Data Set 4), and the results showed good repeatability (Fig. 6A). The peaks from Sample 1 and Sample 2 were mainly located within 1,000-bp upstream of transcription start sites (TSSs) (Fig. 6B). To determine the prospective binding motifs of GhLYI, equally long peak sequences were submitted to MEME-ChIP (http://meme-suite.org/tools/meme-chip) (Brown et al. 2013) and examined for enriched motifs. The only motif that was enriched by MEME tool was ACACGTG (e-value 2.5e−008) (Fig. 6C), and 3 motifs were obtained by STREME tool (Supplemental Fig. S8). In a total of 315 peak sequences of 3 replicates, this motif appeared 76 times, accounting for 24.1%. Also, we performed CUT&Tag assay with transgenic GhLYIOX cottons using FLAG antibody to identify the in vivo binding sites of GhLYI. Totally, 2,005, 1,505 and 1,425 binding peaks respectively in 3 biological replicates were identified and associated with 1,650, 1,190, and 1,168 neighboring genes (Supplemental Data Set 5). There were 489 genes shared among the 3 groups (Fig. 6D). About 17% of the peaks were within 2,000 bp of sequence 5′ to the TSS of a gene (Supplemental Fig. S7). Rather, peaks localized to within 2,000-bp upstream and downstream of TSSs, with the highest frequency being around the TSS itself (Fig. 6E). To identify the actual GhLYI-binding motif, the peak sequences were analyzed using MEME-ChIP too. We analyzed the e-value, concentration, percentage of occurrence, and best matches of these motifs, and some 5- to 7-bp motifs with high confidence and high proportion were shown in Supplemental Fig. S9. Among these motifs, we found the CACGTG motif the similar to Arabidopsis. Also, we found a core sequence GGT/CCC with 3.4e−012 e-value and 9.8% proportion, and there was a clear distribution peak at the center of the input sequences, which can be targeted by TCP transcription factors, such as LFY and TCP16.KEGG pathway analysis was performed with the Dynamic Pathway Enrichment Analysis tool provided by GeneDenovo (https://www.omicshare.com/tools/home/report/koenrich.html) . The target gene sets were found to be mostly enriched for genes associated with photosynthesis, oxidative phosphorylation, RNA polymerase, cutin, suberine, and wax biosynthesis and circadian rhythm (Fig. 6G). In the CUT&Tag experiments conducted with 2 species, we found a common downstream gene GhSAG20, and the enriched peak sequences were very similar. We analyzed the promoter sequence of GhSAG20 using PlantPAN4.0 database (http://plantpan.itps.ncku.edu.tw/plantpan4/index.html) and identified several known plant-binding motifs. Among these motifs, we found that 5′-GGACCg-3′ was consistent with above MEME-ChIP analysis results (Supplemental Table S2). Therefore, we focused on the sequence within GhSAG20 gene promoter region as the site of GhLYI protein binding.

Figure 6.

Genome-wide distribution of GhLYI-binding sites. A) Pearson correlation coefficient analysis of 3 samples. GhLYI-2/3/4 represent 3 biological replicates incubating with GFP antibody in Arabidopsis CUT&Tag experiment; B) distance of GhLYI protein-binding peaks from TSSs throughout the genome. Promoters are defined as −1,000 to +1,000 bp surrounding a TSS; shaded areas around the line represent confidence interval. C) Enriched motif in GhLYI-binding regions in Arabidopsis CUT&Tag experiment. D) Venn diagram shows the overlapped genes among 3 replicates. GhLYI-flag-1/2/3 represent 3 biological replicates incubating with FLAG antibody in cotton CUT&Tag experiment. E) Distribution of genic GhLYI-binding peaks flanking TSSs throughout the genome. Promoters are defined as −2,000 to +2,000 bp surrounding the TSS. The vertical axis represents the coverage depth of reads, the larger the value, the deeper the sequencing depth. F) The enriched motifs identified by MEME-ChIP in the 100-bp flanking sequences around the summits of cotton CUT&Tag peaks. G) KEGG pathway analysis of GhLYI target genes. Only the top 25 significantly enriched (P < 0.05) pathways are shown. Column color indicates significance (adjusted P-value), and the length corresponds to the proportion of genes with the corresponding annotation. Gene percentage: the ratio of the number of DEGs located under the pathway term to the number of all annotated genes located under the pathway term.

GhLYI positively regulates the expression of GhSAG20 by directly binding to its promoter

The binding of GhLYI protein to the promoter of GhSAG20 was firstly verified by yeast 1-hybrid (Y1H) assay. As shown in Fig. 7A, the minimum inhibitory concentration of AbA used to inhibit the growth of yeast Y1H (pAbAi-SAG20) is 100 ng/mL. The yeast cells transformed with GhLYI, and the promoter of GhSAG20 had increased growth ability on SD/-ura-Leu nutritional media with AbA relative to those containing GhLYI and empty vector (Fig. 7A). To verify which region was involved in the combination, we performed a truncation analysis by Y1H additionally. The promoter of GhSAG20 was divided into 3 segments with the length of 16 to 17 bp, and the results showed that the second segment (GCGGAGGACCGTGGAA) could bind to the GhLYI protein (Fig. 7A).

Figure 7.

GhLYI directly regulates the expression of GhSAG20. A) Y1H assay of GhLYI showing its interaction with the peak sequence in the promoter region of GhSAG20. The plasmids PGADT7-GhLYI and PGADT7 were introduced into yeast strain Y1H Gold containing the pAbAi-ProSAG20 vector and cultured on medium without Leu and ura but with AbAi. SAG20-1, SAG20-2, and SAG20-3 represented the 3 segments after further truncation of the GhSAG20 promoter; pAbAi-SAG20: the promoter of GhSAG20 was cloned into pAbAi vector; pGADT7-GhLYI: the CDS of GhLYI was cloned into pGADT7 vector. B) EMSA using the EBS (Probe1) and GhSAG20 peak sequence (Probe2). Arrowheads indicate free probes and protein–DNA complexes. Plus signs indicate presence; minus signs indicate absence. The concentration of biotin-labeled probe in Lane 3 is 3 times that of Lane 2 (right). C) Dual-LUC assay showing GhLYI activation of the GhSAG20 promoter. (Up) Schematics of the reporter construct carrying GhSAG20 promoter fragments and effector constructs carrying GhLYI and empty vector and the transient transcriptional expression analysis showing that GhSAG20 was activated by GhLYI in epidermal cells of N. benthamiana leaves. (Down) The ratio of LUC and REN. Data are mean ± SEM of at least 5 biological replicates. T2G-2 and T2G-45: 2 overexpressed cotton lines with of GhLYI. D) RT-qPCR analysis of the expression level of GhSAG20 in GhLYI overexpression cottons and control materials. Data are means (± Sd) of 3 biological replicates. Double asterisks show significant difference from the mock treatment at P < 0.01 (2-tailed Student's t-test) in Figs. 7C and D.

To further analyze the direct binding of GhLYI to the promoter of GhSAG20, we next examined the binding affinity of purified full-length GhLYI recombinant protein fused with the His tag in EMSA experiments. Before the experiment, the quality of purified protein was tested using western blotting (Supplemental Fig. S10). Our results showed that His-GhLYI could bind the peak sequence in the promoter of GhSAG20 in vitro (Fig. 7B). Specifically, when the 42-bp fragment was used as the probe, biotin-labeled and biotin-unlabeled probes competed efficiently in binding GhLYI. Previous research has reported that EIN3 regulates the expression of its direct target genes by binding with the EBS in their promoters (Solano et al. 1998; Zhang et al. 2018). Therefore, we also designed an EBS probe to detect the ability of GhLYI protein to bind such sequences. No apparent DNA-binding activity was observed, which may be due to GhLYI protein lacking the DBDs that are necessary for interacting with the EBS (Fig. 7B).

Next, to confirm GhLYI mediation of GhSAG20 transcriptional activity, transient expression experiments were performed in which pGhSAG20::LUC as the reporter and 35S::GhLYI as the effector (Fig. 7C) were co-transformed into N. benthamiana leaves. The results showed that co-transfection of GhLYI promoted expression of the luciferase (LUC) reporter gene driven by the peak sequence from the GhSAG20 promoter. Specifically, expression of the reporter construct increased ∼2-fold in the presence of GhLYI (Fig. 7C), which suggested that GhLYI was a facilitator of GhSAG20 transcription. RT-qPCR was performed to detect the transcript level of GhSAG20 in 2 overexpression transgenic cotton lines and TM-1. As expected, the expression level of GhSAG20 was significantly increased in the former (Fig. 7D). Taken together, these results indicate that GhLYI positively regulates the expression of GhSAG20 by directly binding to its promoter.

VIGS of GhSAG20 attenuates leaf senescence

To further determine whether GhSAG20 plays a role in the process of leaf senescence, we applied VIGS in G. hirsutum acc.TM-1 to transiently suppress its expression and observe the resulted phenotypes. About 2 wk after injection, strong photobleaching was present on the newly emerged leaves of seedlings transformed with pTRV2-GhCLA, indicating that the VIGS system was successful under our experimental conditions (Fig. 8A). Similarly, GhSAG20 expression levels were distinctly reduced in pTRV2-GhSAG20 plants as compared to pTRV2 seedlings (Fig. 8B). We then collected leaves of similar size for dark and ACC treatments. Five days later, brown spots began to appear at the edge regions of the leaves in control plants, while the leaves of gene-silenced plants remained completely green. At the seventh day after treatment, the silenced leaves began to show brown spots at their edges, while the brown portion accounted for more than half the leaf area in control leaves. NBT staining revealed fewer blue spots in the leaves of silenced plants, indicating lower content of active oxygen (Fig. 8C). Collectively, these results suggest that knockdown of GhSAG20 attenuates leaf senescence.

Figure 8.

Silencing of GhSAG20 attenuates leaf senescence. A) Phenotype of pTRV2-GhCLA at 2-wk post-Agrobacterium infiltration. Scale bar, 2 cm. pTRV2-GhCLA: cloroplastos alterados 1 gene sequence was cloned into pTRV2 vector as positive control. B) Expression of GhSAG20 in pTRV2-GhSAG20 and control plants. Data are means (± Sd) of biological replicates and error bars indicate SEM. Double asterisks show significant differences from the mock treatment at P < 0.01(2-tailed Student's t-test); pTRV2-1/2/3: empty vector was used as negative control; pTRV2-SAG20-1/2/3/4/5/6:GhSAG20 sequence was cloned into pTRV2 vector. C) Senescence phenotypes in detached leaves of GhSAG20-silenced plants and control plants after dark and ACC treatment. Scale bar, 1 cm. pTRV2: empty vector as control; pTRV2-GhSAG20:GhSAG20 sequence was cloned into pTRV2 vector causing the silencing of GhSAG20.

Discussion

The truncated EIL protein GhLYI accelerates leaf senescence by regulating the transcription of SAG20

The EIN3/EIL gene family is a small group of transcription factors in vascular plants. The proteins encoded by these genes range from 185 to 896 aa in length (Mao et al. 2022). Here, we found that GhLYI encodes a substantially shorter protein that only retains the BD I and BD II within the DBD, proving that these domains are the most conserved aspect in the gene structure of the EIN3/EIL family. In this work, through genetic transformation of Arabidopsis, we proved that the truncated EIL protein GhLYI also plays a role in ethylene signal transduction.

Leaf senescence, a process of programmed cell death, has been shown in Arabidopsis to be promoted by various phytohormones such as ethylene, abscisic acid, and JA (Ueda et al. 2020; Zhang et al. 2020; Yu et al. 2021b). Ethylene influences leaf senescence, in which effect EIN3/EIL plays the role of core transcriptional factor. EIN3 binds directly to the promoter of miR164 and inhibits its expression, thereby indirectly promoting the expression of ORESARA1 (ORE1), a core regulator of leaf senescence (Li et al. 2013). In addition, EIN3 promotes senescence by directly inducing the expression of CCGs such as NYE1, NYC1, and PAO (Qiu et al. 2015). Additionally, EIN3 physically interacts with NPR1, together synergistically promoting the transcriptional activity of ORE1 and SENESCENCE-ASSOCIATED GENE 29 (SAG29) in senescing leaves (Wang et al. 2021a) (Fig. 9). Here, we identified that GhLYI acts as a positive regulator of senescence progression. Firstly, the transcription level of GhLYI increases with aging and with dark treatment, a condition used to quickly induce nonnatural leaf senescence. Secondly, both ectopic expression of GhLYI in Arabidopsis and its overexpression in cotton accelerate leaf senescence, with more rapid loss of chlorophyll and earlier induction of SAG expression. Thirdly, RNA-seq profiling analysis of cotton overexpressing GhLYI revealed that most of the upregulated DEGs are enriched in plant hormone signal transduction, plant–pathogen interaction, starch and sucrose metabolism, and fatty acid degradation, while those downregulated relate to photosynthesis, flavonoid biosynthesis, and biosynthesis of secondary metabolites. Finally, CUT&Tag assays helped us to identify the SAG GhSAG20 as a direct target of GhLYI, which interaction we then demonstrated by EMSA, Y1H, and LUC experiments. Based on these data, we proposed a GhLYI-GhSAG20 model summarizing the role of GhLYI in accelerating leaf senescence.

Figure 9.

The proposed model for AtEIN3 and GhLYI modulation of the senescence process. EIN3 participates in a coherent feed-forward loop regulating ethylene-mediated leaf senescence. EIN3 physically interacts with the core SA signaling regulator NPR1 in senescing leaves. EIN3 and NPR1 then synergistically promote expression of the senescence-associated genes ORE1 and SAG29. EIN3 also directly represses the transcription of miR164, which negatively regulates ORE1 at the posttranscriptional level. Meanwhile, EIN3 and ORE1 share 3 common direct targets: NYE1, NYC1, and PAO, which contribute to leaf senescence. GhLYI is an important ethylene signaling pathway transcription factor in cotton whose transcription can be induced by age and dark treatment. GhLYI could directly bind to the promoter of GhSAG20 and positively regulate its expression to accelerate the aging process. Arrows and bars represent positive and negative regulation, respectively, and 2-way arrows represent interaction.

In maize (Zea mays), soybean (Glycine max), rice (O. sativa), and wheat (Triticum aestivum), it has been estimated that if the life span of functional leaves were prolonged during the grain filling stage, crop production would improve by 1% to 2% (Kamal et al. 2019; Ostrowska-Mazurek et al. 2020; Lee and Masclaux-Daubresse 2021). We found that GhLYIOX transgenic cottons showed a phenotype of reduced plant height, shortened stems, premature leaves, smaller flower organs, and extremely high bud falling rate. We speculate that premature senescence of these plants leads to a large number of flower buds falling off, thus reducing the number of bolls, which impacts their yields. All told, the findings of this study provide fundamental insights into the role of this unique truncated EIL gene in cotton, which will support future functional examination of the various important biological molecular mechanisms fulfilled by this vital gene family.

The truncated EIL protein still retains DNA-binding activity

Sequence-specific DNA-binding activities have been demonstrated for the proteins AtEIN3, AtEIL1, AtEIL2, and Tobacco EIN3-like (TEIL). In particular, a series of deletion mutants were tested for DNA binding, and the N-terminal half except for the first ∼80 residues was found to be indispensable (Solano et al. 1998). Song et al. (2022) similarly identified the functional region of the transcription-activating domain in BpEIN3.1 to be among its C-terminal region, with the 519 to 538 aa region being especially required. However, the full-length aa sequences encoded by GhLYI contain neither the DBD nor the transcription-activating domain that have been reported. Nonetheless, our binding experiments in vitro demonstrated that the truncated GhLYI protein still has DNA-binding activity and moreover recognizes a binding site different from the previously found EBS motif.

CUT&Tag is a method that replaces the traditional ChIP-seq to study the interaction between a protein and DNA and determine DNA-binding sites. In the original papers relating to this technique, only histone modification, NPAT, and CCCTC-binding factor (CTCF) were tested (Hyle et al. 2019; Kaya-Okur et al. 2019; Akdogan-Ozdilek et al. 2022). Many transcription factors are not rich in expression, bind DNA only weakly or temporarily, or bind indirectly to chromatin. In these cases, chromatin cross-linking and ultrasound are necessary steps to detect protein DNA interactions (Ouyang et al. 2021). In this paper, we successfully used Arabidopsis and cotton leaves to investigate the DNA-binding sites of GhLYI. In addition to the SAG20 gene mentioned in this article, our CUT&Tag results also included a cell wall modification-related gene, specifically a xyloglucan endotransglucosylase/hydrolase (XTH22), for which we verified the binding of GhLYI protein to its promoter by Y1H and EMSA methods (Supplemental Fig. S11). Together, these findings prove the reliability of our CUT&Tag results.

As GhLYI protein completely lacks the C-terminal region containing the known activating domain, we further explored the mechanism of its regulatory role by transforming a pGBKT7 vector containing the CDS of GhLYI into yeast 2-hybrid (Y2H) Gold yeast cells. Yeast co-transformed with both the pGBKT7-GhLYI plasmid and pGADT7 grew well on SD/-Trp-Leu medium but not on SD/-Trp-Leu-His medium, proving GhLYI had no transcriptional activation activity (Supplemental Fig. S12). The CUT&Tag technology helped us find a GhLYI-binding motif which contained the analogous DOF TFs binding site. DOFs have also been shown to participate in leaf senescence. For example, AtDOF2.1 has been reported to play an active role in JA-induced leaf senescence through the MYC2-DOF2.1-MYC2 feed-forward transcription loop (Zhuo et al. 2020). In rice, OsDOF24 delays leaf senescence by suppressing the activity of the OsAOS gene related to JA biosynthesis (Shim et al. 2019). Like DOF proteins, GhLYI may need to interact with other TFs to ensure the precise targeting and binding of DNA and subsequently promote transcription. Based on previous studies, we know that A. thaliana EIN3/EIL family proteins function as homodimers and speculated that GhLYI may affect the normal function of other EIN3 proteins (Solano et al. 1998; Wang et al. 2021b). Accordingly, Y2H assays were applied to test the interaction between GhLYI and other EIN3/EIL family members in upland cotton. We randomly chose 4 EIN3/EIL proteins and introduced their coding regions into the pGADT7 vector. Yeast clones containing pGBKT7-GhLYI + pGADT7-other EIN3/EIL did not grow normally on SD/-Trp-Leu-His medium (Supplemental Fig. S13), indicating that GhLYI did not interact directly with those proteins. In 2003 and 2010, Guo et al. confirmed that EIN3 and EIL1 can interact with 2 F-Box like proteins EBF1/EBF2 and be degraded through the ubiquitination pathway (Guo and Ecker 2003; An et al. 2010). Other studies have shown that the main transcription inhibitor JAZ and the important transcription factor MYC2 in the JA signaling pathway, as well as the important inhibitor DELLA in the gibberellin signaling pathway, can directly interact with EIN3 to inhibit its transcription activity (Zhu et al. 2011; An et al. 2012; Song et al. 2014). EIN3 also physically interacted with the core SA signaling regulator NPR1 in senescing leaves. They synergistically promoted the expression of the SAGs ORE1 and SAG29 (Wang et al. 2021a). Qiao et al. reported that a transcriptional repressor of EIN3-dependent ethylene-response 1 (TREE1) interacts with EIN3 to regulate transcriptional repression that leads to an inhibition of shoot growth (Wang et al. 2020). In order to explore how GhLYI performs its regulatory function, we identified the genes with the highest similarity to AtEBF1/2, AtMYC2, AtDELLA, and AtNPR1 protein sequence in upland cotton and utilized the Y2H system to verify whether they can interact with GhLYI. Unfortunately, we had not found any interaction between GhLYI and the above genes (Supplemental Fig. S14). The basic/helix–loop–helix (bHLH) proteins are a superfamily of eukaryote transcription factors whose N-terminal alkaline region contains ∼15 aa, including 6 alkaline aa. This region is involved in DNA binding and can identify E-box (5′-CAGCTG-3′) and G-box (5′-CACGTG-3′), thereby regulating gene transcription (Atchley et al. 1999). In the CUT&Tag experiment, we found the same cis-element and speculated that it may be due to the similar alkaline aa regions retained in the protein structure of the truncated GhLYI gene. However, we did not observe the binding of GhLYI protein and G-box probe in the EMSA experiment. The reason for this result may be that the binding of proteins to DNA not only depends on sequence specificity but may also be influenced by the number, arrangement, and flanking nucleotides of the hexanucleotide core (Massari and Murre 2000). In addition, we all know that the nucleosome of eukaryotic cell is the core structural element of chromatin, which is composed of DNA and 5 histones (H1, H2A, H2B, H3, and H4).The structure of chromatin changes dynamically. DNA replication and transcription need the opening of the tight structure of chromatin to allow trans-acting factor to bind with DNA. Histone modification, DNA methylation, and nucleosome remodeling all affect the chromatin accessibility (Klemm et al. 2019). We speculate that in addition to binding with specific motifs, GhLYI may also control gene expression from an epigenetic level. Although further biochemical study is required to reveal the function and regulation mechanism of the truncated EIL protein, the results of this study provide insights that will aid future studies of the mechanisms underlying the EIN3-mediated senescence process as well as plant growth and development.

Materials and methods

Plant materials and treatment

Arabidopsis (A. thaliana) ecotype Col-0 is the parent line for all mutants and transgenic plants used in this study. Seeds were surface sterilized with 80% (v/v) alcohol for 10 min, rinsed with sterile water 4 times, and then plated on MS agar plates (half-strength MS, 1% [w/v] phyto agar). After stratification at 4 °C for 3 d, the seedlings were transferred to soil and grown at 22 °C under long-day conditions (16-h light/8-h dark). For ethylene treatment, 5 or10 μM ACC (an ethylene precursor) was added into the MS agar plates. After 3 d of stratification at 4 °C in the dark, the plates were wrapped in foil and kept at 22 °C in an incubator; seedling phenotypes were analyzed after 3 further days. For the senescence experiments, plants were grown under either long-day conditions (16-h light/8-h dark) or short-day conditions (8-h light/16-h dark). For the dark treatment, the third and fourth leaves were detached from 4-wk-old plants and incubated in a box with constant high humidity in the dark at 22 °C.

Upland cotton (G. hirsutum L. acc. TM-1) was also used in this study. Cotton seedlings were grown in pots at 28 °C in a greenhouse with a 16-h light/8-h dark cycle and 60% humidity. Ethylene treatment was applied to 4-wk-old cotton seedlings by watering and spraying the leaves with 10 μM ACC. Samples were taken at 0, 1, 3, 6, 12, and 24 h after treatment; pure water was used as control. For hormone- and dark-induced leaf senescence, the first true leaves from a 4-wk-old cotton plant were detached and floated on 50 mL of distilled water supplemented with 10 µM ACC and kept in the dark at 22 °C.

Leaf senescence assays

Chlorophyll measurement: Chlorophyll content of the third and fourth Arabidopsis rosette leaves was measured according to an established protocol (Zhang et al. 2015b). Chlorophyll pigments were extracted with 80% (v/v) ice-cold ethanol from leaf tissues of plants transferred to a dark environment for the indicated time. Extracts were centrifuged at 12,000 × g for 10 min at 4 °C, and the absorbance at 645 and 663 nm was determined using a UV-vis spectrophotometer. Six individual leaves of each genotype were measured, and 3 biological replicates were performed. DAB and NBT staining: The third and fourth leaves of 18-d-old plants were vacuum infiltrated with DAB tetrahydrochloride solution (1 mg/mL DAB and 0.1% [v/v] Tween 20 in 10 mM Na₂HPO₄) and NBT solution (0.5 mg/mL NBT, 10 mM potassium phosphate, pH 7.8, and 10 mM sodium azide) to detect hydrogen peroxide and superoxide, respectively. The leaves were incubated in the dark for 8 to 10 h, and decolorized in 95% ethanol (v/v) to remove the chlorophyll. The intensity of brown and blue coloration indicates H₂O₂ and ${\mathrm{O}}_2^{-}$ content, respectively. Images of the leaves were captured using a digital camera. All staining experiments were repeated 3 times, and representative results were presented in the figure. Take 10 to 12 leaves from each experimental group and control group for staining in each experiment.

Phylogenetic analysis

Arabidopsis EIN3 proteins were used to search possible cotton EIN3/EIL sequences by BLASTP (Altschul et al. 1997) according to a method previously applied in G. arboreum L. (Zhou et al. 2014), G. raimondii L. (Wang et al. 2012), G. hirsutum L. (Zhang et al. 2015a), and Gossypium barbadense L. (Wang et al. 2019). The genome sequences of G. hirsutum (TM-1 ZJU v2.1), G. barbadense (Hai7124 ZJU v1.1-a1), G. arboretum (A2 CRI-updated_v1), and G. raimondii (D5 JGI_v2_a2.1) were downloaded from COTTONGEN (http://www.cottongen.org). Clustal X was used to align EIN3 protein sequences. MEGA X was used to align the aa sequences of EIN3/EIL proteins to construct an unrooted phylogenetic tree (Kumar et al. 2018). A neighbor joining (NJ) consensus tree was constructed with the following parameters: P-distance, pairwise gap deletion, and bootstrap (1,000 replicates). The average expression levels and numbers of expressed genes in 35 vegetative and reproductive tissues of G. hirsutum L. acc. TM-1 was from our previous report (Zhang et al. 2015a).

Reverse transcription quantitative PCR

Total RNA was extracted from cotton leaves using the EASYspin Plus Plant RNA kit (Molfarming, RK16-50T). Reverse transcription was performed using HiScript II Q RT SuperMix for RT-qPCR (Vazyme, R223-01). The obtained cDNA was diluted 1:10 and subjected to quantitative PCR using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) and StepOne & StepOnePlus Real-Time PCR Systems (Thermo Fisher Scientific) according to the manufacturer's manual. The following PCR program was used: 95 °C for 2 min, followed by 40 cycles at 95 °C for 15 s, 55 °C for 15 s, and 72 °C for 15 s, and finished with a melting-curve analysis. Histone3 was adopted as an internal control. Primers used for RT-qPCR are listed in Supplemental Data Set 1. All experiments are repeated 3 to 5 times, and each biological replicate are represented by 3 technical replicates.

Transcriptome analyses

We collected leaves and young buds from transgenic cottons line T2G-45 for RNA-seq analysis. Total RNA was extracted using the same procedure as for RT-qPCR analysis. Sequencing was performed with the Illumina HiSeq 4000 platform, and the obtained clean reads were mapped to the reference genome of G. hirsutum (TM-1_V2.1) with Hisat2 (version 2.2.0). Transcript expression was evaluated by StringTie v1.3.5 (Pertea et al. 2015), and DEGs were identified using featureCounts Version 2.0.0 (Liao et al. 2014). DEGs were selected using Student's t-test with P < 0.05.

Cleavage under targets and tagmentation

CUT&Tag was performed as described previously (Tao et al. 2020). Briefly, hyperactive pG-Tn5/pA-Tn5 transposase was made following the manual of the CUT&Tag kit (Vazyme, cat. no. S602/S603). 1.5- to 2-g fresh plant materials were fixed with 1% (v/v) formaldehyde for 15 min immediately before nuclear extraction. Nuclear extracts were incubated with 1 μL of antibody (anti-GFP antibody; Abcam, ab290), anti-FLAG antibody (Thermo, MA191878), or IgG control antibody, diluted 1:50 to 1:100; the final concentration of antibody was 10 to 20 μg/mL) overnight at 4 °C with gentle shaking. Experiments performed with 3 biological replicates. Guinea pig antirabbit secondary antibody (Novus Biologicals, NBP1-72763) was used to amplify the signal. Transposase was added and immunoprecipitation allowed to proceed for 1 h at room temperature with gentle shaking, after which Mg²⁺ was added and the mixture incubated at 37 °C for 1 h to activate the transposase. DNA successfully cut and ligated with the linker was extracted, and the library was established by PCR (TAE, Vazyme). The purified product was sent to Novogene for next-generation sequencing using an Illumina HiSeq 2500, which yielded 6 to 7 Gb of raw data. Clean reads were aligned to the A. thaliana or G. hirsutum genome using Hisat2 with default parameters (Kim et al. 2015). Peak calling was performed using MACS2 (v2.1.7), and the resulting output files were visualized using deeptools2 (Ramirez et al. 2016). A gene was regarded as GhLYI-bound if its promoter region (including 2 kb upstream of the TSS) had at least 1 bp overlapping any peak. To discover conserved binding motifs, the 100-bp sequence of binding peaks was submitted to MEME-ChIP. For functional category analysis, KEGG pathway information was collected from the KEGG database and functional categories from the MapmanWeb site. The enriched functions of genes bound by GhLYI were determined using OmicShare tools, a free online platform for data analysis (http://www.omicshare.com/tools). The calculated P-value was subjected to FDR correction, taking FDR ≤ 0.05 as the threshold for significance.

Y1H and Y2H assays

Y1H assays were performed according to the manufacturer's instructions (Clontech Mountain View, CA, USA) (Zhu et al. 2022). In brief, the pGADT7 vector was linearized by EcoRI and BamHI, and pAbAi vector was linearized by HindIII and SmaI; the CDS of GhLYI and peak sequences obtained by CUT&Tag were respectively cloned into the pGADT7 and pAbAi vectors. The pAbAi vectors harboring constructs were linearized by digestion with BstBI and integrated into the genome of the Y1H Gold strain. Yeast cells were grown at 30 °C for 3 d on SD/-ura selection plates representing a gradient of aureobasidin A (AbA) concentrations. pGADT7-GhLYI or empty pGADT7 vector was subsequently transformed into the yeast strain containing the pAbAi constructs. Yeast cells were grown for 3 d on SD/-ura-Leu selection plates with minimum inhibitor concentration. Y2H assays were performed according to the manufacturer's instructions (Clontech Mountain View, CA, USA). In brief, the CDS of GhLYI and speculative interacting genes were respectively cloned into the pGBKT7 and pGADT7 (linearized by SmaI and BamHI) vectors and co-transformate into Y2H Gold strain. Yeast cells were grown at 30 °C for 3 d on SD/-Trp-Leu selection plates. Positive monoclonal cells were grown for 3 d on SD/-Trp-Leu-His selection plates.

Dual-LUC transient expression assay

Bioluminescence assays were performed as described previously (Song et al. 2018). pGreen II-62-SK-GhLYI and pGreen II-62-SK were used as effector constructs and pGreen II-0800-LUC vector containing pGhSAG20::LUC as the reporter construct. The prepared suspensions were injected into N. benthamiana leaves and grown for 3 d. The leaves were then sprayed with 5 mM luciferin and kept in the dark for 5 min to quench the fluorescence. The experiments were repeated 3 times, and representative photo was showed in the figure. Subsequently, LUC activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA). Infected leaf areas were placed in liquid nitrogen and then ground into fine powder, of which 100 mg was suspended in 100-µL protein extraction buffer in a 1.5 mL tube, mixed completely, and placed on ice for 20 min. Tubes then were centrifuged at 12,000 × g and 4 °C for 15 min, after which the supernatant containing crude proteins was transferred to a new tube. Next, 20 µL of crude protein extract was mixed with 100-µL LAR II in a 96-well microplate, and the firefly LUC activity was measured on a Microplate Reader (H1MD Take3 Trio). Afterward, 100-µL Stop & Glo reagent was added to each well, and the Renilla (REN) LUC activity was measured. Promoter activity was calculated as the ratio of LUC to REN. Experiments were repeated 3 times, and at least 5 biological replicates were set for experimental group and corresponding control.

Electrophoretic mobility shift assay

Purified GhLYI-His recombinant protein was used for EMSA; this protein was generated by introducing the coding region of GhLYI into the pET-30a vector at the BamHI and HindIII restriction sites. GhLYI-His was expressed in Escherichia coli strain BL21(DE3) and then purified for use in EMSA. Biotin-labeled/biotin-unlabeled oligonucleotide probes were synthesized by Tsingke Biotechnology Co., Ltd; their sequences are listed in Supplemental Data Set 1. EMSA was performed using the EMSA/Gel-Shift Kit (Beyotime, GS002) according to the manufacturer's instructions. Probes were obtained by annealing the biotin-labeled WT or mutant complementary oligonucleotides. Briefly, 3 μg of GhLYI-His fusion proteins was incubated together at room temperature for 20 min with biotin-labeled WT and cold competitor probes in 20-μL reaction mixtures and then separated on 6% native polyacrylamide gels.

VIGS assays

To further study whether knockdown of GhSAG20 affected leaf senescence, we conducted VIGS experiments. Briefly, a 208-bp fragment near the TSS of GhSAG20 (A10) was amplified from cDNA using the VIGS primers. The pTRV2 empty vector was digested with the EcoRI and BamHI restriction enzymes, and the cloned sequence fragment was inserted using homologous recombinase, after which it was transformed into E. coli. The positive recombinant plasmid was subsequently transferred into Agrobacterium tumefaciens GV3101, which was then formulated as an infection solution. Cotton seedlings with 2 fully expanded cotyledons but without a true leaf were selected for injection using a needle-less syringe. After injection, the plants were left in a culture room under darkness overnight and subsequently grown at 23 °C under long photoperiod (16/8 d/night). Plants were divided into the following 3 groups: (i) a positive control group, in which pTRV2-GhCLA and pTRV1 empty vectors were mixed at a 1:1 ratio and injected into the plants; (ii) a negative control group, in which pTRV2 and pTRV1 empty vectors were mixed at a 1:1 ratio and injected into the plants; and (iii) the experimental group, in which pTRV2-GhSAG20 and pTRV1 empty vectors were mixed at a 1:1 ratio and injected into the plants. When the second true leaves on pTRV2-GhCLA positive control plants developed photobleaching, silencing efficiency was tested using RT-qPCR with primers SAG20-A10-DL-F/R.

Accession numbers

Arabidopsis sequence data in this article can be found in the Arabidopsis Information Resource (TAIR, http://www.Arabidopsis.org/index.jsp) under the following accession numbers: AtEIN3 (AT3G20770) and AtSAG12 (AT5G45890). G. hirsutum sequence data can be found in the COTTONGEN (http://www.cottongen.org) data libraries under accession numbers GhLYI (Gh_D08G0312), GhSAG20 (Gh_A06G1407), GhSAG12 (Gh_A05G1308), GhNAP (Gh_A12G1505), and GhWRKY53 (Gh_D08G1232).The data sets presented in this study can be found in online repositories. The accession numbers are given below: BioProject PRJNA917110, PRJNA954143 and PRJNA917109.

Acknowledgments

We thank Prof. Hongwei Guo for providing the ein3eil1 mutant seeds.

Author contributions

T.Z.Z. conceived, supervised, and provided funding for the subject. Y.Y.Z. performed most of the experiments and wrote the article. Y.H.Z., H.Y., and Z.Y.Z. provided advice on experimental design and writing. J.W.C. and S.L.F. provided advice on bioinformatics data analysis.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1. Phylogenetic tree of 3 different cotton species and Arabidopsis EIN3/EIL proteins.

Supplemental Figure S2. Comparison of the sequence after stop codon of the GhLYI CDS region and other EIN3/EIL gene sequences.

Supplemental Figure S3.GhLYI gene knockout materials have no phenotypes in leaf senescence

Supplemental Figure S4. Validation of RNA-seq results by RT-qPCR.

Supplemental Figure S5. PCA and Pearson correlation analysis of RNA-seq data.

Supplemental Figure S6. Nucleus extraction and constructed library gel map from the CUT&Tag assay.

Supplemental Figure S7. Distribution of GhLYI-binding peaks in gene region categories.

Supplemental Figure S8. The enriched motifs of Arabidopsis CUT&Tag peaks by MEME-ChIP STREME tool.

Supplemental Figure S9. Motif enrichment analysis in cotton CUT&Tag peaks.

Supplemental Figure S10. Protein purification and western blot of GhLYI protein.

Supplemental Figure S11. Y1H assay and EMSA assay show GhLYI binds to the promoter sequence of XTH22.

Supplemental Figure S12. Y2H assay between GhLYI protein and other EIN3/EIL1 proteins.

Supplemental Figure S13. Y2H assay between GhLYI protein and GhEBF1, GhNPR1, GhDELLA, and GhMYC2 proteins.

Supplemental Table S1. The counts of EIN3/EIL family members and corresponding protein length ranges in 8 species

Supplemental Table S2. Analysis of the GhSAG20 promoter sequence using PlantPAN3.0 database

Supplemental Data Set 1. Primers used in this study

Supplemental Data Set 2. DEGs in the leaves of GhLYI overexpression transgenic cotton plants, identified by RNA-seq

Supplemental Data Set 3. DEGs in 0 dpa ovules of GhLYI overexpression transgenic cotton plants, identified by RNA-seq

Supplemental Data Set 4. Genome-wide binding sites and target genes of GhLYI in Arabidopsis, identified by CUT&Tag

Supplemental Data Set 5. Genome-wide binding sites and target genes of GhLYI in cotton, identified by CUT&Tag

Funding

This study was financially supported by grants from the NSFC (32130075, 32260179), the Leading Innovative and Entrepreneur Team Introduction Program of Zhejiang (2019R01002), the Fundamental Research Funds for the Central Universities (226-2022-00100), and China Postdoctoral Science Foundation (2022M712813).

Data availability

All relevant data are within the manuscript and its additional files.

Author notes

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/General-Instructions) is Tianzhen Zhang ([email protected]).

Conflict of interest statement. None declared.