(2022) 22:248
Zhou et al. BMC Plant Biology
https://doi.org/10.1186/s12870-022-03622-9

Open Access

RESEARCH

Overexpression of AHL9 accelerates leaf
senescence in Arabidopsis thaliana
Yusen Zhou1,2†, Xiaomin Zhang1,2†, Jing Chen1,2†, Xiaopeng Guo1,2, Hongyan Wang1,2, Weibo Zhen1,2,Junli Zhang1,2, Zhubing Hu1,2, Xuebing Zhang1,2, José Ramón Botella3, Toshiro Ito4* and Siyi Guo1,2*

Abstract
Background: Leaf senescence, the final stage of leaf growth and development, is regulated by numerous internal
factors and environmental cues. Ethylene is one of the key senescence related hormones, but the underlying molecular mechanism of ethylene-induced leaf senescence remains poorly understood.
Results: In this study, we identified one AT-hook like (AHL) protein, AHL9, as a positive regulator of leaf senescencein Arabidopsis thaliana. Overexpression of AHL9 significantly accelerates age-related leaf senescence and promotes
dark-induced leaf chlorosis. The early senescence phenotype observed in AHL9 overexpressing lines is inhibited by the
ethylene biosynthesis inhibitor aminooxyacetic acid suggesting the involvement of ethylene in the AHL9-associatedsenescence. RNA-seq and quantitative reverse transcription PCR (qRT-PCR) data identified numerous senescenceassociated genes differentially expressed in leaves of AHL9 overexpressing transgenic plants.
Conclusions: Our investigation demonstrates that AHL9 functions in accelerating the leaf senescence process via
ethylene synthesis or signalling.
Keywords: Arabidopsis thaliana, AT-hook like proteins, Ethylene, Leaf senescence, Transcription regulation
Background
Senescence is an intricate and highly orchestrated process in the plant’s life cycle. A range of biological eventsoccur at the physiological, biochemical, and molecularlevels during this period, including chloroplast degradation, hydrolization of macromolecules, reduction ofcytoplasmic volume, and decrease in cellular metabolic
activity. The most noticeable feature in leaf senescence isthe rapid degradation of chlorophyll during chloroplastdisassembly, which leads to the yellowing of leaves [1].
†Yusen Zhou, Xiaomin Zhang and Jing Chen are contributed equally to thiswork.
*Correspondence: itot@bs.naist.jp; guosiyi@henu.edu.cn

1State Key Laboratory of Crop Stress Adaptation and Improvement, Schoolof Life Sciences, Henan University, Kaifeng 475004, China
4
Division of Biological Science, Nara Institute of Science and Technology,
Ikoma, Nara 630‑0192, Japan
Full list of author information is available at the end of the article

During senescence, leaf nutrients are remobilized andrelocated from the dying leaves to seeds or other storage
tissues, thereby contributing to the fitness and survivalof plant [2–4]. Although senescence is an active process
to relocate nutrients from old tissues, precocious senescence will shorten the growth stage of crops and result in
reduced yield and crop quality [2, 5, 6].
Senescence involves massive transcriptional changes,
and a large number of senescence-associated genes(SAGs) have been identified. The expression of SAGs isregulated by senescence-related transcription factors TFs
such as NACs (NAM [No Apical Meristem], ATAF2[Arabidopsis Transcription Activation Factor2] andCUC2 [Cup-shaped Cotyledon2]), MYBs, bZIPs (basic
region/leucine zipper motifs) and WRKYs TFs [7–10].
Incubation of detached leaves in the dark is highly effective for induction of SAGs, leaf yellowing and chlorophyll
loss. Therefore, it has been widely used as a model system
for the study of leaf senescence [11].

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Zhou et al. BMC Plant Biology(2022) 22:248

Although leaf senescence occurs in an age-dependent
manner, the process is also greatly affected by multiple
endogenous and environmental signals coordinating thelife span of leaves to optimize plant fitness. Endogenous
signals such as plant hormones play vital roles in thesenescence process via complex interconnecting pathways. Positive regulators include abscisic acid, ethylene,
jasmonic acid, salicylic acid, brassinosteroid and strigolactone, in contrast, cytokinin, gibberellic acid and auxin
suppress senescence [12, 13]. Ethylene has long been
established as a key hormone regulating the timing andprogression rate of leaf senescence [14, 15]. In vitro application of ethylene induces premature leaf senescence,
while application of inhibitors of ethylene biosynthesis or
action can delay leaf senescence symptoms [16, 17].
Transcriptional analysis has shown that the expression
levels of a number of genes encoding ethylene biosynthesis, such as 1-aminocyclopropane-1-carboxylic acidsynthase (ACS) and ACC oxidase (ACO), and signaling
components increase in senescence leaves [16], indicating ethylene signaling is involved in the regulationof senescence leaves. This is further supported by theextended leaf longevity of ethylene-insensitive mutants
that are defective in ethylene signaling transduction, suchas etr1 (ethylene resistant 1), ein2 (ethylene insensitive2) and ein3 [18–20]. To date, the regulation of ethylene
seems to be achieved by the EIN2-EIN3-miR164-NAC2
signalling cascade. EIN2 regulates miR164 expression, as well as its downstream target gene ORESARA1(ORE1) ORE1/NAC2 [21]. EIN3 acts at the downstreamof EIN2 to promote chlorophyll degradation by affecting chlorophyll catabolic genes [22]. Although the ethylene biosynthetic pathway and downstream key elements
involved in ethylene signal transduction have been extensively studied through genetic approaches, the transcriptional network leading to leaf senescence remains largely
unknown.
AT-HOOK MOTIF CONTAINING NUCLEAR
LOCALIZED (AHL) proteins are transcription factors
featured with two conserved structural units: a plant andprokaryote conserved (PPC) domain, involved in protein–protein interactions, and one or two DNA-binding
AT-hook motif(s) [23–27]. The AT-hook motif contains
a conserved palindromic core sequence, Arg-Gly-Arg,
capable to bind to the minor groove of AT-rich B-form
chromosomal DNA, thus changing its architecture andcontrolling the expression of corresponding genes [25,
28–30]. AHL family proteins have been proposed toregulate plant growth and development. AHL22 acts asa chromatin remodeling factor that regulates FT (FLOWERING LOCUS T) expression to promote flowering[31]. Several AHLs are involved in hormonal homeostasis and response, especially gibberellins, cytokinins andPage 2 of 12

jasmonic acid [28, 32, 33]. ESC/AHL27 (ESCAROLA)
and SOB3/AHL29 (SUPPRESSOR OF PHYTOCHROME
B-4 #3) act redundantly to repress hypocotyl elongationin response to light [24, 34]. Overexpression of AHL27 in
Arabidopsis delays senescence and increases post-harvest
storage life, but the molecular mechanism is unclear [29].
Recently, it has been reported that SOB3/AHL29 repress
petiole growth by antagonizing PIF-mediated transcriptional activation of genes associated with growth andhormonal pathways [35].
Although the fact that one member of the AHL clade A
is involved in leaf senescence, the detailed mechanism bywhich AHL transcription factors regulate leaf senescencestill remain largely unknown. Here, we identified AHL9
as a positive regulator of leaf senescence in Arabidopsis. The overexpression of AHL9 causes a severely earlysenescence phenotype. Moreover, we showed in the current study that AHL9 not only promotes dark-induced
but also ethylene-induced leaf chlorosis. Our RNA-seq
analysis showed that the expression of multiple SAGs isaltered in AHL9 overexpressing lines. Our results provide new insights into the molecular mechanism of leaf
senescence highlighting AHL9 as a prominent regulatorycomponent in dark-induced and ethylene-induced leaf
senescence.

Results
Overexpression of AHL9 results in premature leaf
senescence

AHL family proteins include clade A and clade B subfamilies [24, 26]. To further explore the functions of AHL
clade B subfamily, a phylogenetic tree was generated andanalysed (Fig. S1). AHL9 was chosen to study its biological functions. Bioinformatics analysis demonstrates that
AHL9 genome fragments contain 5 exons and 4 introns,
the AHL9 protein has two AT-hook motifs and one
DUF296 (plant and prokaryote conserved (PPC)/domainof unknown function #296 domain) (Fig. S2). AHL11 is
the closest homolog of AHL9 in Arabidopsis genome,
which exhibits 62.2% sequence identity with AHL9 basedon full-length alignment (Fig. S3). Through the quantitative reverse transcription PCR (qRT-PCR) assay, various
expression abundancy of AHL9 was detected. However, ahigher expression in the aging tissues (the fourth and fifth
rosette leaves) than in proliferative tissue (the siliques)
was observed, indicating AHL9 may have the role during the senescence process (Fig. S4). To test the biological function of AHL9, 35S::AHL9 transgenic Arabidopsis
lines were generated. Two independent transgenic lines(35S::AHL9-OE10 and OE11) were further analysed.
Compared with WT, both transgenic lines displayedpremature leaf senescence at 32 d (Fig. 1A). In addition
to precocious leaf senescence, the rosette leaves of both

Zhou et al. BMC Plant Biology(2022) 22:248

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Fig. 1 Overexpression of AHL9 causes early senescence in rosette leaves. A Leaf phenotypes of 32-d-old WT, AHL9-OE10 and AHL9-OE11
overexpressing plants. Scale bar = 1 cm. B Leaves detached from (A) and arranged according to their age. Rosette leaves were numbered frombottom to top with the first leaf being the oldest. Scale bar = 1 cm. C Representative images of the fourth and fifth rosette leaves detached from
WT, AHL9-OE10 and AHL9-OE11 plants of the different ages. D Chlorophyll contents of the leaves shown in (C). *P < 0.05, **P < 0.01, Data indicate
means ± SD, n = 3. Statistical analyses were performed using one-way ANOVA. Means with different letters above the bars indicate statisticallysignificant results (P < 0.05). The experiment was conducted three times with similar results

AHL9 transgenic lines became elongated and narrow(Fig. 1A). To gain a better view of the function of AHL9
in leaf senescence, the rosette leaves of transgenic lines
were compared with their corresponding WT at the samestage. As shown in Fig. 1B, when the first five leaves of
AHL9-OE10 and AHL9-OE11 had already turned yellow,

only the first three leaves became yellow in WT. In contrast with an apparent premature senescence of AHL9transgenic lines, the AHL9 T-DNA insertion mutant
was almost like the WT (Fig. S5). Phylogenetic analysis showed that AHL9 hold the similarity with AHL11,
AHL5 and AHL12, indicating the possible functional

Zhou et al. BMC Plant Biology(2022) 22:248

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redundancy. As AHL11 exhibited the highest similarity
with AHL9, we generated the double mutants that lackof both AHL9 and AHL11. All plants (WT, ahl11, ahl9
ahl11-1 and ahl9 ahl11-2) exhibited the similar rate inleaf senescence, implying the higher-order redundancy
among the proteins belonging to AHLs family. (Fig. S6).
To further analyse the development of age-related
senescence in WT and AHL9 overexpressing plants, thefourth and fifth rosette leaves detached from WT and
35S::AHL9 transgenic lines were used for further analysis
in the different stages. At 20 and 26 d after germination,
there was no significant difference in leaf colour between
transgenic lines and WT. However, at 32 d after sowing,
the fourth and fifth rosette leaves of 35S::AHL9 transgenic lines became yellow, while the same stage leaves of
WT were still green. The yellowing of these leaves progressed rapidly in the AHL9-OEs transgenic lines, andthe majority of them become completely yellow at 38 d
after germination, at this stage, WT plants only started toshow signs of leaf senescence with only the tip of the leaf
turning yellow (Fig. 1C). This is consistent with previous
reports showing that senescence symptoms usually startfrom the tip and outer edge of a rosette leaf at a given age[36]. Chlorophyll quantification assays confirmed the visual phenotypic observations showing significantly lower
chlorophyll content in the fourth and fifth rosette leaves
in AHL9 overexpressing transgenic lines in 32 d and 38
d-old plants, compared with WT plants (Fig. 1D).

(AOA), an ethylene biosynthesis inhibitor, respectively.
As expected, pre-treatment with ACC increased senescence in WT and 35S::AHL9 leaves (Fig. 2C, D). In contrast, the enhanced senescence observed in 35S::AHL9
leaves was suppressed by treatment with ACC and AOA(Fig. 2C, D), suggesting that the role of AHL9 in senescence is dependent on ethylene. Unlike ethylene, ABA
treatment did not significantly increase the differenceof chlorophyll content between WT and AHL9 overexpression transgenic lines (Fig. S7). These results indicate the involvement of AHL9 in ethylene-induced leaf
senescence.

AHL9 regulates dark‑ and ethylene‑induced leaf
senescence

In order to investigate the possible roles of AHL9 inleaf senescence, we performed genome-wide expression profiling of AHL9-OEs and WT plants under normal growth conditions. Leaves from 30-d old WT andboth AHL9-OEs transgenic lines were used for RNAseq experiments. In total 20 million uniquely mapped
reads per sample with high reproducibility among all
three biological replicates were generated (Table S1).
Analysis of AHL9-OE10 vs WT identified 953 genes
with statistically significant differences in gene expression ­(log2(fold_change) > 1, q-values < 0.01), including 342
up-regulated and 611 down-regulated genes (Fig. 4A-B
and Table S2). In the case of AHL9-OE11, 1451 genes
showed statistically significant differences with WT, with
556 up-regulated and 895 down-regulated genes. Whenboth datasets are put together, a common set of 665 differentially expressed genes (DEGs) were identified in
AHL9-OE10 and AHL9-OE11 vs WT (Fig. 4C). Interestingly, some of the genes with altered expression belong
to NAC, WRKY, IAA, and AP2 transcription factors,
ACC oxidase, and the ETHYLENE RESPONSE SENSOR(Fig. 4D). Principal component analysis (PCA) and correlation analysis of all datasets indicated strong RNA-seq
reliability (Fig. S8-S9). qRT-PCR analysis of 10 randomly

Incubation of detached leaves in darkness is often used
as an effective method to stimulate synchronous senescence [37]. To further probe the potential roles of AHL9
in leaf senescence, we examined dark-induced leaf senescence in three-week-old WT and 35S::AHL9 transgenic
plants. Rosette leaves of the same age detached from
3-week-old WT and 35S::AHL9 plants were similar incolour before dark treatment; however, after 3 days darkness, 35S::AHL9 leaves exhibited stronger leaf chlorosis
than WT (Fig. 2A). The phenotypic observations wereconfirmed by chlorophyll assays showing a more pronounced decrease in chlorophyll levels in the AHL9-OE
leaves compared to WT (Fig. 2B), suggesting that darkinduced leaf senescence is promoted by the overexpression of AHL9.
Phytohormones play critical roles in leaf senescence,
therefore we queried whether the overexpression of
AHL9 altered the ethylene- and ABA-induced senescence. For this purpose, we first examined the darknessinduced senescence of detached leaves upon treatment
with either the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or ACC plus aminooxyacetic acid

AHL9 is a nuclear localized AT‑hook protein

To establish the AHL9 subcellular localization, an AHL9GFP fusion construct or GFP (control) were transiently
expressed in Arabidopsis mesophyll protoplasts under
the control of the cauliflower mosaic virus 35S promoter. The nuclear marker protein H2B-mCherry was
co-expressed to visualize nuclei [38]. In the GFP controls, green fluorescence was observed throughout the
protoplasts (Fig. 3). Green fluorescence in AHL9-GFP
transfected protoplasts was restricted to the nuclei andoverlaped with the yellow fluorescence of the nuclear
marker, indicating that AHL9-GFP is located in the
nucleus (Fig. 3).
Identification of differentially expressed genes in AHL9
overexpression lines

Zhou et al. BMC Plant Biology(2022) 22:248

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Fig. 2 AHL9 is involved in dark-induced and ethylene-induced leaf senescence. A Phenotype of detached leaves from 3-week-old WT and two
independent AHL9 overexpressing lines subjected to dark treatment. Detached leaves were incubated in MES buffer for 3 d under dark conditions.
Scale bar = 1 cm. B Chlorophyll content of detached leaves from (A). The data were analyzed using one-way ANOVA analysis. Means with different
letters above the bars indicate statistically significant results (P < 0.05). Data indicate means ± SD, n = 3. The experiment was conducted three times
with similar results. C Phenotype of detached leaves from 3-week-old WT and two independent AHL9 overexpressing lines ACC or ACC + AOA in thedark. Detached leaves were treated with MES buffer, 100 µM ACC or 100 µM ACC + 500 µM AOA for 3 d under the dark conditions. D Chlorophyll
contents in leaves from (C). The data were analyzed using one-way ANOVA analysis. Means with different letters above the bars indicate statisticallysignificant results (P < 0.05). Data indicate means ± SD, n = 3. The experiment was conducted three times with similar results

Fig. 3 AHL9 is localized exclusively in the nucleus. Arabidopsis protoplasts were co-transfected with expression cassettes containing either 35S::GFP
and 35S::H2B-mCherry or 35S::AHL9-GFP and 35S::H2B-mCherry. GFP signals were detected using a laser confocal scanning microscopy. H2B-mCherry
was used as a nuclear marker. From left to right are green fluorescence signal, nuclear marker, chlorophyll red auto fluorescence, bright-field andmerged images, respectively. Scale bars = 10 μm

Zhou et al. BMC Plant Biology(2022) 22:248

chosen DEGs are consistent with the RNA-seq results(Fig. 4E, Table S3). Gene Ontology analysis highlighted
the expression changes of genes involved in response tostimuli, hormone response, biological process and biological regulation (Fig. 5). Given that gene regulatory
networks composed of interactions between TFs (transcription factors) and their targets have been implicatedin controlling leaf senescence, we conducted GO enrichment analysis for these TFs. Indeed, we found most ofthese TFs were significantly enriched in GO terms that
may associate with biological process, such as the RNA
biosynthetic or metabolic process, cellular macromolecule biosynthetic process and nitrogen compound metabolic process (Fig. S10). These results provided molecularevidence supporting premature leaf senescence in AHL9
overexpression lines.

Discussion
In recent years, our knowledge about the molecularmechanisms triggering leaf senescence has expanded
significantly. Transcriptomic analysis of leaf senescencerevealed the expression changes of thousands of SAGs,
however, only a small portion of them have been functionally characterized [8]. Besides, factors on the top of
the regulation module regulating diverse SAGs and/or
other functional genes have also been identified througheither loss-of-function and/or gain-of-function studies inmodel plants such as Arabidopsis and rice [2, 23]. Amongthese regulators, transcription factors are interesting
candidates as they can influence the expression of multiple genes during the senescence process.
The study of AHL family genes revealed different roles
in plant growth and development, such as hypocotyl
elongation, flower development, gibberellin biosynthesis
and leaf longevity [25, 28, 31, 34]. Although knowledge
about the functional role of AT-hook motif proteinsis still very limited in plants. Notably, Ectopic ORE7/
ESC delays leaf senescence, which probably up-regulate
genes that suppress senescence and down-regulate genesthat enable the progression of the senescence process
through modification of chromatin architecture[29]. In
this study, we identify and characterize an AHL protein,
AHL9, involved in leaf senescence. Overexpression of
AHL9 results in early senescence in Arabidopsis (Fig. 1).
Transgenic plants overexpressing AHL9 also exhibited

Page 6 of 12

accelerated dark-induced leaf senescence (Fig. 2A). In
contrast, the lack of senescence-related phenotype in
the ahl9 mutant may be due to the existence of functionally redundant genes, since 4 paralogs were identified in
Arabidopsis, including AHL5, AHL12, AHL9, and AHL11[26]. The lack of phenotype in the ahl9 mutant could also
be explained by functional compensation from othersenescence-associated pathways, since leaf senescence is
the integrated result of various pathways that incorporate
numerous endogenous factors and environmental signals[8, 23]. Taken together, these results suggest that AHL9
behaves like an early senescence-activator that influences
both age and dark-induced leaf senescence.
Although ethylene has been known for many decades
to be a senescence-inducing plant hormone, the molecular mechanism underlying ethylene-mediated senescenceremains largely unknown. Comparative transcriptome
analyses have revealed a number of ethylene biosynthesis and signalling genes, with elevated transcript levels insenescing leaves, supporting the idea that sensitivity of aleaf to ethylene might account for the age-dependent leaf
senescence [16, 18]. Here, we show that AHL9 is involvedin the control of leaf senescence through ethylene synthesis or signalling since pre-treatment with the ethylene
biosynthesis inhibitor AOA, can effectively repress theincreased senescence observed in AHL9 overexpressing
transgenic lines. Consistent with this data, some ethylene synthesis and signalling genes are induced in the
RNA-seq data, although the genes with altered expression may not be the direct targets of AHL9 (Fig. 4D).
Intriguingly, we also find some of these TFs that show
DEGs are significantly enriched in GO terms that may
associated with stress response such as response to abiotic stimulus, ethylene-activated signaling pathway andhormone-mediated signaling pathway. Ethylene is sensed
by a five-member family of ethylene receptors on theendoplasmic reticulum [39], then this binding inactivates a Raf-like Ser/Thr kinase, CTR1 (CONSTITUTIVE
TRIPLE RESPONSE1), thereby releases the C-terminalend of the positive regulator, EIN2, to the nucleus andstabilizes EIN3 and ETHYLENE INSENSITIVE3-LIKE1(EIL1), which in turn, activate the expression of ethylene
target genes to promote premature senescence in leaves[40–43]. Identifying specific target genes and interaction
proteins of AHL9 in vivo will be the next step required to(See figure on next page.)
Fig. 4 RNA-seq analysis of WT, AHL9-OE10 and AHL9-OE11 transgenic lines. A-C Diagram showing differentially expressed genes (DEGs). DEGs wereclassified according to their expression fold-changes (FC) in the pairwise genotypic comparison between AHL9-OE10 or AHL9-OE11 and WT leaves(q-value < 0.01, |log2(fold change)|> 1). D Heatmap of senescence-associated genes (SAGs) in DEGs of AHL9-OE10, AHL9-OE11 and WT. The color bar
indicates the normalized gene expression. E Expression levels of ten randomly chosen DEGs identified in the RNA-seq experiments. qRT-PCR was
performed using cDNA from 30-d old leaves of AHL9-OE10, AHL9-OE11 and WT. qRT-PCR values are expressed as the mean ± SD compared to that of
the internal control (UBQ10). The fold change of the qRT-PCR was determined by the efficiency method (­ 2−ΔΔCT). Error bars indicate SD. n = 3, t-test,**P < 0.01. Assays were done in triplicate

Zhou et al. BMC Plant Biology(2022) 22:248

Fig. 4 (See legend on previous page.)

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Zhou et al. BMC Plant Biology(2022) 22:248

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Fig. 5 Gene ontology analysis of DEGs. Gene ontology terms of DEGs identified in the RNA-seq experiments. A-B AHL9-OE10 vs WT (A), and (B)AHL9-OE11 vs WT. CC, cellular component. MF, molecular function. BP, biological process

gain a more in-depth knowledge of its role in regulating
leaf senescence.

Conclusions
The current study demonstrates that one AT-hook protein, AHL9, may regulate leaf senescence via the finetuning of ethylene biosynthesis or signalling.
Methods
Plant materials and growth conditions

The Arabidopsis thaliana ecotype Columbia (Col-0) wasused in this study [44]. The transfer DNA (T-DNA) insertional mutant ahl9 (GK_735D06) was obtained from theNottingham Arabidopsis Stock Centre (NASC). Seeds
were surface sterilized in 10% (v/v) sodium hypochlorite
for 10 min, washed 3 times with sterilized water, and thengrown on Murashige and Skoog medium plus 3% sucrose
and 0.6% agar (pH 5.8) after 2 d vernalization in darkness
at 4℃. The 7-d-old seedlings were transferred into soil
and were grown at 22℃ in a 16-h-light/8-h-dark cycle for
additional experiments and seed production. Yusen Zhou
and Jing Chen undertook the formal identification of theplant materials (T-DNA mutants, CRISPR-Cas9 mutants
and transgenic plants) used in this study.
Plasmid construction and plant transformation

AHL9 cDNAs were obtained by RT-PCR of RNA isolated from Col-0 seedlings, the full length of AHL9 CDS
was amplified by PCR method using primers. According to the instructions of the invitrogen gateway kit(kit No.11789 (BP Clonase); No.117910 (LR Clonase)),the PCR products were cloned into pDNOR221 vector
using the Gateway™ BP Clonase™ II Enzyme mix. Subsequently, AHL9 CDS was sub-cloned into the pDEST
Gateway binary vector pGWB405 between the 35S promoter and GFP gene to produce the 35S::AHL9-GFP
construct. The constructed plasmids were introduced
into Agrobacterium tumefaciens strain GV3101 andtransformed into plants using the floral dip method [45].
Transgenic plants in the ­T2 generation with T-DNA
insertion at a single locus were selected by kanamycin
resistance, and ­T3 homozygotes were used for all analyses. These primers were listed in Table S3.

Zhou et al. BMC Plant Biology(2022) 22:248

Generation of ahl11 and ahl9 ahl11 CRISPR‑Cas9 mutants

For AHL11 CRISPR-Cas9 mutant generation, first, thesgRNAs of AHL11 were designed using the CRISPOR
online internet (http://​crisp​or.​tefor.​net/), we selected
two sgRNAs (T1sgRNA: GAG​GAG​GAG​GAC​CAG​AAC​
CG, T2sgRNA: ACA​GAC​GTG​TCA​CCT​GAA​GG). Second, two sgRNAs were constructed into pCAMBIA1300pYAO-cas9 vector [46]. Finally, the constructed vectors
were introduced into WT and ahl9 mutants using theAgrobacterium mediated floral dip method respectively.
The stable inheritance CRISPR-Cas9 mutants were confirmed through PCR and Sanger sequencing.
Dark‑induced senescence assay

For the dark-induced senescence assay, the fourth andfifth rosette leaves were carefully detached from 3-weekold soil-grown Arabidopsis. Detached rosette leaves wereincubated on MES buffer (0.5 × MS, 3 mM MES, pH 5.8)
in complete darkness for 3 d and sampled for analyzing
leaf senescence and chlorophyll content.
Hormone induced leaf senescence assay

The fourth and fifth leaf of 3-week-old plants weredetached and floated on 3 mL of MES buffer (0.5 × MS,
3 mM MES, pH 5.8) supplemented with or without ABA(10 μM, 50 μM, 100 μM, and 200 μM), 1-aminocyclopropane-1-carboxylic acid (ACC, 100 μM) and 100 μM ACC
plus ethylene biosynthesis inhibitor (AOA, 500 μM). The
petri dishes were sealed with parafilm tape and wrappedwith double-layer aluminum foil, then the petri dishes
were put in a black box to avoid light. All hormone treatments were performed at 22℃ under the dark conditions.
Three biological replicates were performed.
Determination of chlorophyll content

Chlorophyll was measured according to the method
described by Li et al. [47]. Briefly, Arabidopsis seedlings
were weighed (W), placed into Eppendorf tubes withacetone (95%, 1 mL, V) and kept overnight under dark
conditions. Samples were then centrifuged at 13,000 g
for 10 min and absorbance values at 665 nm and 649 nm
were obtained from the supernatant. Chlorophyll content(including chlorophyll a and b) was calculated according to the following formula: chlorophyll a = 13.95A665– 6.88A649; chlorophyll b = 24.96A645 – 7.32A665; andtotal chlorophyll = (chlorophyll a + chlorophyll b) × V/W.
At least three independent samples were examined, allof which produced the typical results reported in thisarticle.

Page 9 of 12

described previously [48]. Transformed protoplasts wereobserved using a fluorescence microscope (Ziess confocal LSM710). H2B-mCherry were used as controls fornuclear localization [38].
qRT‑PCR analysis

Total RNA was isolated from the seedlings with Trizol
reagent and DNA was digested by RNase-free DNase
I. Two µg of total RNA was used for reverse transcription with M-MLV reverse transcriptase according to the
supplier’s instructions (Promega). qRT-PCR analyses
were performed with Roche Light Cycler 480 real-time
PCR system using the SYBR Green Master Mix (Vazyme
Biotech Co., Ltd.) and specific primers for PCR amplification. UBQ10 was used as an internal control for datanormalization. These primers were listed in Table S3.
RNA‑seq analysis

The sixth, seventh and eighth rosette leaves were individually collected from 30-d-old WT, AHL9-OE10 and
AHL9-OE11, pooled, and frozen in liquid nitrogen. The
samples were stored at − 80℃ prior to RNA extraction. The RNA-seq analysis was performed at the Berry
Genomics Corporation (Beijing) with three biologicalreplicates. Briefly, The cDNA library were constructed
and sequenced on HiSeq 2000 sequencing system. The
clean data was produced by discarding the paired reads
that one read’s number of N base is more than 5 or has
more than 30% bases with a low quality value below 15.
The clean reads were aligned to the reference genomeof Vigna angularis using Hisat2 [49]. Then samtools andHTSeq-count were used to count the reads number of
each gene and gene’s expression level was normallized
as Fragments Per Kilobase Million (FPKM) [50]. DESeq2
was used to analyze differential gene expression, basedon the negative binomial distribution, of the replicate
samples between the treatment group and control group[51]. A threshold value of p-adjusted value (qvalue) < 0.01(|log2foldchange|> 1) was used to obtain differentiallyexpressed genes (DEGs). The gene ontology (GO) enrichment, with p-adjusted value cut-off 0.05, were performed
using custom R scripts based on Bioconductor packages
goseq and GO.db in order to classify DEGs into terms.
Phylogenetic tree

Phylogenetic tree was generated using MEGA-X software. The AHL clade B family from Arabidopsis thaliana,
some orthologs of rice and maize protein sequences were
downloaded from NCBI.

Subcellular localization

Statistical analysis

The 35S::AHL9-GFP and 35S::GFP plasmids were transformed into Arabidopsis mesophyll protoplasts as

All experiments were repeated with at least three times.
The presented data were expressed as the means ± SD.

Zhou et al. BMC Plant Biology(2022) 22:248

Statistical analyses were performed using one-way
ANOVA or Student’s t-test.
Accession numbers

The Arabidopsis Genome Initiative identifiers for the genes
described in this article are as follows: AHL9 (At2g45850),
AHL11 (At3g61310), LOX1 (AT1G55020), IAA1
(AT4G14560), UBQ10 (AT4G05320), ACO2 (AT1G62380),
NAC079 (AT5G07680), anac048 (AT3G04420), ERF039(AT4G16750), PCAP2 (AT5G44610), SAUR72 (AT3G12830),
LTP4 (AT5G59310), UGT85A1 (AT1G22400), PAO4
(AT1G65840), WRKY63 (AT1G66600), EBF2 (AT5G25350),
NAC003 (AT1G02220), NAC100 (AT5G61430), AP2(AT4G36920), ERS1 (AT2G40940), AHL2 (AT4G22770),
SAUR41 (AT1G16510), IAA7 (AT3G23050), IAA29(AT4G32280), IAA14 (AT4G14550), IPT3 (AT3G63110).

Page 10 of 12

S6 Generation and phenotypic analysis of ahl11 and ahl9 ahl11 double
mutants. A Schematic genomic structure of AHL11 and the sgRNA target
sites. Black boxes indicate exons, lines indicate introns, empty boxes indicate
5’UTR and 3’UTR of AHL11. UTR, untranslated region. B Sanger sequencing of
the ahl11, ahl9 ahl11-1 and ahl9 ahl11-2 mutants in WT and ahl9 backgrounds. C Amino acids of AHL11, ahl11, ahl11-1, and ahl11-2. D Phenotypic
analysis of WT, ahl9, ahl11, ahl9ahl11-1 and ahl9ahl11-2 plants grown for
15-d, 21-d, 28-d and 34-d. Scale bars = 2 cm. Fig. S7 The senescencephenotypes of detached leaves from AHL9 overexpressing plants treatedwith ABA. Detached leaves were treated with MES buffer (Mock), 10 μM, 50
μ M or 100 μM and 200 μM ABA for 3 d under dark conditions. Fig. S8 PCA
analysis of RNA-seq of AHL9-OE10 vs WT, AHL9-OE11 vs WT. PCA results forthe transcript-level (FPKM) dataset of all samples. The sample with samecolor means samples from the same group. Fig. S9 Correlation analysis of
the samples from the same group. R­ 2 represents correlation coefficient. Fig.
S10 Gene ontology analysis of different expression transcription factors.
Additional file 2 Table S1 RNA-seq maped reads of WT, AHL9-OE10, andAHL9-OE11 leaf samples.
Additional file 3 Table S2 Differentially expressed genes of WT, AHL9OE10, and AHL9-OE11.
Additional file 4 Table S3 Primers used in this study.

Abbreviations
AHL: At-Hook motif containing nuclear Localized; SAG: Senescence-Associated Gene; CUC​: Cup-shaped Cotyledon; NAC: Nam, Ataf2 and Cuc2; ACC​
: 1-Aminocyclopropane-1-Carboxylic acid; ACS: ACC synthase; ACO: ACC
oxidase; AOA: ACC plus aminooxyacetic acid; ETR1: Ethylene Resistant 1; EIN2:
Ethylene Insensitive 2; PPC: Plant and Prokaryote Conserved; DUF296: Domainof Unknown Function #296 domain; DEGs: Differentially Expressed Genes;
PCA: Principal Component Analysis; TF: Transcription Factor; EIL1: Ethylene
Insensitive3-Like1; GO: The Gene Ontology.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12870-​022-​03622-9.
Additional file 1 Fig. S1 Phylogenic analysis of AHLs clade B family proteinsin Arabidopsis thanalia and orthologs in Oryza sativa L. and Zea mays L..
The protein sequences were downloaded from NCBI database (https://www.ncbi.nlm.nih.gov/). And then the phylogenetic tree was generatedusing MEGA-X software. The numbers of the branches are the bootstrap
values from 1,000 replicates. Fig. S2 Schematic diagrams of AHL9 genomic
and protein structure. A Genomic structures of AHL9. Arrows represent
start codon. Black boxes represent exons. Crease lines represent introns.
White boxes represent 5’ and 3’ untranslation regions. Scale bar, 100 bp. B
Protein structures of AHL9. Red boxes represent AT-hook motif. Gray boxes
represent DUF296 domain. Fig. S3 Protein sequences alignment of AHL9
and AHL11. The AHL9 and AHL11 sequences were downloaded from NCBI
database (https://www.ncbi.nlm.nih.gov/). The fasta format was generatedusing CLUSTALW software, and then the alignment was produced with
GENEDOC software. Fig. S4 qRT-PCR analysis of AHL9 transcript levels inroots, leaves, stems, inflorescences, and siliques in six-week-old plants. The
roots, leaves, stems, inflorescences, and siliques were sampled from three
individual plants at different development stages, respectively. The fourthand fifth rosettes were used to determine the gene expression in the leaves.
Actin was used for normalization purposes. Data indicate mean ± SD (n =3). Three biological repeats were performed. Fig. S5 Phenotypic observationof WT and ahl9 plants. A Schematic structure of AHL9 and the positionsof the T-DNA insertion in ahl9. Black boxes indicate exons, lines indicate
introns, empty boxes indicate upstream and downstream regions of AHL9.
Triangles represent the T-DNA insertion. B Amplification of AHL9 in genomic
DNA from the WT and ahl9. LP and RP primers were used for AHL9 genomic
sequence amplification. LB and RP primers were used for specific amplification of the T-DNA insertion. C qRT-PCR detection of AHL9 transcript in
28-d-old WT and ahl9. UBQ10 was as an internal control. Three biologicalrepeats were done. **P ˂ 0.01, Student’s t-test. D-E Phenotypic observation of 28-d (D) and 36-d (E) old WT and ahl9 plants. Scale bars, 1 cm. Fig.

Acknowledgements
We appreciate members of Prof. Chun-Peng Song’s group in Henan University
for participating in helpful discussions. No conflicts of interest declared.
Authors’ contributions
S.G. designed the study. Y.Z. and J.C. undertook the formal identification of theplant material (T-DNA mutants, CRISPR-Cas9 mutants and transgenic plants)
used in this study. Y.Z., J.C., X.G., H.W., and J.Z. performed the experiments.
W.Z., Z.H., and Xue.Z. carried out the re-annotation and comparative analyses.
Xiao.Z., J.R.B., T.I., and S.G. analyzed the data and wrote the manuscript. All
authors read and approved the final manuscript.
Funding
This work was supported by grants from the Natural Science Foundation ofChina (31671517 and 31970808), and the Program for Innovative Research
Team (in Science and Technology) in University of Henan Province (21IRTSTHN019) to S.G., the 111 Project#D16014 to J.R.B., and a JSPS KAKENHI
Grant-in-Aid for Scientific Research A (20H00470), and a Grant-in-Aid forchallenging Exploratory Research (No. 21K19266) to T.I.. The funding bodies
had no role in the design of the study, data analysis, interpretation and writing
the manuscript.
Availability of data and materials
The RNA-seq data have been deposited into sequence read archive (SRA)
database under the Bioproject PRJNA780875. The BioProject’s metadata isavailable at https://​datav​iew.​ncbi.​nlm.​nih.​gov/​object/​PRJNA​780875?​revie​
wer=​sjfjt​gdgtd​4alus​r7nf9​l5has3 in read-only format. All relevant data andplant materials are available from the corresponding authors upon request.

Declarations
Ethics approval and consent to participate
All methods were performed in accordance with the relevant guidelines andregulations.
Consent for publication
Not applicable.
Competing interests
The authors declare that there are no conflicts of interest.
Author details
1State Key Laboratory of Crop Stress Adaptation and Improvement, Schoolof Life Sciences, Henan University, Kaifeng 475004, China. 2 State Key
Laboratory of Cotton Biology, School of Life Sciences, Henan University,

Zhou et al. BMC Plant Biology(2022) 22:248

Kaifeng 475004, China. 3 Plant Genetic Engineering Laboratory, School of Agriculture and Food Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia. 4 Division of Biological Science, Nara Institute of Scienceand Technology, Ikoma, Nara 630‑0192, Japan.
Received: 14 November 2021 Accepted: 28 April 2022

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