TaMIR5086, a
microRNA Member in Triticum aestivum, Confers Plants Drought Tolerance via Modulating Stomata Movement and
Antioxidant Enzyme Activities
Yingjia Zhao1,2†, Menghao Zhao1,2†, Guiqing Shi1,2, Lan Wang1,2, Chenyang Ni1,2, Zehui Wang1,2, Ying Zhang1,2, Han Xue1,2 and Kai Xiao1,2*
1State Key
Laboratory of North China Crop Improvement and Regulation, Baoding, P. R. China
071001
2College of
Agronomy, Hebei Agricultural University, Baoding, Hebei 071001, P. R. China
*For correspondence: xiaokai@hebau.edu.cn
†Contributed equally to this work and are
co-first authors
Received
15 March 2021; Accepted 07 June 2021; Published 18 September 2021
Abstract
The microRNA (miRNA) members exert essential roles in
regulating the growth and development of plants as well as plant response to
various abiotic stresses. In this study, TaMIR5086, a member of the miRNA
family in wheat (Triticum aestivum L.), was
subjected to functional evaluation in plant drought tolerance. TaMIR5086 has
six target genes that are involved in transcription, translation, protein
degradation, and trafficking. The transcripts of TaMIR5086 were elevated in
both aerial and underground tissues within a 48-h regime of drought treatment.
Moreover, the drought-induced expression levels of which were restored
following a normal recovery progression. In contrast to TaMIR5086, all the
target genes reversed the expression modes upon drought stress. These results
suggested that these target genes are regulated under control of TaMIR5086 at
posttranscriptional level. Transgene analysis on TaMIR5086 validated its
positive function in regulating drought stress adaptation; the transgenic lines
overexpressing TaMIR5086 displayed improved plant growth, biomass, and
photosynthetic function compared with wild type under drought treatment. Moreover,
the stomata closing rates were promoted and the activities of superoxide
dismutase (SOD), catalase (CAT) and peroxidase (POD) were enhanced in the
drought-treated transgenic lines. These results suggested that TaMIR5086 improves
drought adaptation by regulating stomata movement and AE catabolism. TaSOD3, TaCAT5 and TaPOD9, the
genes in AE families, modified transcription in the drought-challenged
transgenic lines, suggesting their contribution to reactive oxygen species (ROS)
homeostasis and drought adaptation via
modifying AE activities underlying the miRNA regulation. Therefore, TaMIR5086
acts as one useful index in evaluating drought adaptation and molecular
breeding of drought-tolerant cultivars in T.
aestivum. Our findings provide insight into understanding of drought-tolerant
mechanisms underlying modulation of distinct miRNA members. © 2021 Friends Science
Publishers
Keywords: Wheat (Triticum aestivum L.); miRNA; Drought
stress; Plant growth; Physiological traits; Antioxidant enzymes
Introduction
Drought stress
negatively impacts on the plant growth, development and yield formation for
cereal crops (Cattivelli et al. 2008). Recent investigations have
documented that water deficit leads to modification of numerous physiological
processes, which are associated with root development, cellular membrane
integrity, photosynthetic pigment metabolism, osmolytes biosynthesis, and
tissue and organ establishment (Sallam et
al. 2019). The alterations of them finally result in deterioration of plant
growth traits and the yield formation potential (Benjamin and Nielsen 2006; Seleiman et al. 2021). Therefore, adoption of drought-tolerant varieties has
been an effective strategy in enhancing crop productivity and water utilization
ability of the cereal crops cultivated under water-saving conditions.
Under drought conditions, plants acclimate to the adverse
stressor through various mechanisms evolved at physiological and molecular
levels (Wang et al. 2016; Seleiman et al. 2021). Thus far, the molecular
networks underlying plant drought adaptation have been extensively
investigated. A quantity of regulators has been identified to modulate the
biochemical pathways associated with transduction of the drought signaling.
Among them, the protein receptors, mitogen-activated protein kinases (MAPK),
calmodulin-binding proteins, phosphatases, and distinct transcription factors,
act as the critical components in the drought signal transduction processes and
regulate largely the drought response (Shinozaki and Yamaguchi-Shinozaki 2007; Mittal et
al. 2017; Jagodzik et al. 2018;
Cui et al. 2019). These findings
suggested the complicate nature of drought signal transduction in plants and
distinct regulatory components are valuable in efforts for generating the
drought-tolerant crop germplasms.
The microRNA (miRNA) members
constitute a large class of non-coding RNA in plant species. With nucleotide acids from 20 to
24 nt in length, the miRNA members are involved in the regulation of quantities
of physiological processes, based on their roles in controlling the target
mRNAs at posttranscriptional or translational level via a base pairing mechanism (Zhang et al. 2006; Lu et al.
2011). Thus far, it has been documented that miRNA members are functional in mediating
diverse biological processes, such as growth phase transition, senescence,
floret tissue establishment and root architecture establishment (Rubio-Somoza
and Weigel 2011; Swida-Barteczka and Szweykowska-Kulinska 2019; Zhao et al. 2019). In addition, distinct members in plant miRNA family
have also been recorded to impact on plant responses to abiotic stress
conditions (Ji et al. 2018; Liu et al. 2019; Nadarajah and Kumar 2019).
For example, miR319 in Arabidopsis
responds to dehydration, salt, and chilling stresses at transcriptional level
(Lv et al. 2010; Thiebaut et al. 2012). Functional characterization on this miRNA member
indicated that it conferred plants increased adaptation to above stressors, via modulating transcription of the
transcription factor (TF) genes in TCP family (for TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING
CELL FACTORS [PCF]) that
encode basic helix-loop-helix (bHLH) transcription factors (Ori et al. 2007; Nag et
al. 2009). Meanwhile, an aliquot of the miRNA members in cereal crops
sensitively respond to the drought stress (Nadarajah and Kumar 2019). Rice
miR393 has been documented to regulate plant development and adaptation to
multiple abiotic conditions (Zhao et al.
2019). Thus far, although the relations between plant drought response and
miRNA members are largely established, the mechanisms underlying miRNA-mediated
drought acclimation are needed to be further characterized in plant species,
especially in cereal ones.
As one of the important cereal crops, wheat (T. aestivum) is widely cultivated and contributes
greatly to food security worldwide. However, a large quantity of water
consumption is needed for wheat cultivation due to the long growth period
together with less precipitation in most cultivation regions, which results in
shortage of the water resource and limitation to the sustainable crop
production. Therefore, improving WUE for wheat plants is an urgent issue for a
long term of crop production. Thus far, a quantity of the miRNA family members
of T. aestivum has been stored in the
miRNA bank (www.mirbase.org). Moreover, a suite of investigations concentrated
on characterizing the miRNA functions and predicting the corresponding target
genes in T. aestivum species have
also been documented (Sun et al.
2014; Wang et al. 2014; Bakhshi et al. 2017). However, the mechanisms
underlying the miRNA-mediated physiological processes and stress responses in
this species are largely unknown and need further elucidation. In this study,
TaMIR5086, a miRNA member in wheat, was subjected to functional evaluation for the
role in regulating plant drought response. Our investigation aimed at
elucidating the drought-tolerant mechanisms mediated by miRNA members and
providing essential regulators for generating crop germplasms to be drought defensiveness.
Materials and
Methods
Characterization
of TaMIR5086
Ourexpression evaluation on the miRNA members derived
from wheat (Trtitcum aestivum L.) revealed
that TaMIR5086 (accession number MI0017949) displayed upregulated transcripts
upon drought stress (our unpublished data), which suggested the involvement of
it in drought response at transcriptional level. The precursor sequence,
stem-loop feature, and mature sequence of TaMIR5086 were derived from the miRNA
database of T. aestivum
(www.mira.org).
Evualution
of the target genes of TaMIR5086
The genes interacted by TaMIR5086
were identified based on an online tool
(psRNATarget, Plant microRNA Potential Target Finder; http://plantgrn.noble.org/psRNATarget/).
The cDNA databases of T. aestivum
used for scanning the target genes were Triticum aestivum (bread wheat), cDNA,
EnsemblPlant, release 43.
Putative roles of the target genes were predicted by performing BLASTn analysis
supplemented in NCBI, using target cDNA sequences as queries.
Determination of transcripts
of TaMIR5086 and its target genes
The transcripts of TaMIR5086 and its target genes in
response to drought condition were determined based on qRT-PCR. With this aim,
wheat (cv. Shimai 22) seedlings were cultured in a standard Murashige
and Skoog (MS) solution under following condition: photoperiod of 12 h/12 h
(day/night) with 300 μmolE·m-2·s-1
light intensity during light phase, temperature of 28°C/23°C (day/night), and
relative humidity from 65 to 70%. They were then subjected to the simulated
drought treatment by growing in a MS solution containing PEG-6000 (10% concentration,
w/v). At times of 6, 12, 24 and 48 h under drought treatment, the underground
and aerial organs were sampled. In addition, an aliquot of the 48 h-treated
seedlings were transferred to MS solution for a normal recovery treatment. The
tissues mentioned were sampled at 6, 12, 24 and 48 h under the recovery
condition. The tissues collected prior to simulated drought treatment (referred
to as 0 h) were used as control. qRT-PCR for the samples was conducted
following the previous method (Guo et al. 2013). In brief, total RNA in samples was
isolated using TRIzol reagents (Invitrogen, USA). After removal of putative
genomic DNA using DNase (TaKaRA, Dalian, China), total RNA (~2 μg)
in each sample was used for synthesis of cDNA using the RT-AMV transcriptase
(TaKaRa, Dalian, China). qRT-PCR reaction was conducted using following
components: 12.5 μL of SYBR Premix ExTaqTM (TaKaRa, Dalian, China),
0.5 μL of forward and reverse primers, 1 μL cDNA and
10.5 μL nuclease-free water. The transcripts of TaMIR5086 and its
target genes were determined following the 2-ΔΔCT method, with wheat constitutive gene Tatubulin
to normalize the target miRNA and its target genes (Wang et al. 2020). The primers used for TaMIR5086 and its targets are
shown in Table S1.
Evaluations of growth feature, dry mass and
photosynthetic parameters in transgenic lines
The lines with TaMIR5086 overexpression were established
for determining function of this miRNA in mediating the drought response. With
this aim, the precursor sequence of TaMIR5086 was RT-PCR amplified with
specific primers (Table S1) and inserted into binary expression vector referred
to as pCAMBIA3301 controlled underlying the CaMV35S promoter. The binary
cassette was then integrated into A. tumefaciens (strain EHA105) using
the conventional heat-shock approach and subjected to genetic transformation
onto T. aestivum (cv. Shimai 22) as
previous description (Guo et al. 2013).
Two lines at T3 generation with more TaMIR5086
transcripts (Fig. S1), OE 2 and OE 3 together with wild type (WT), were used to
characterize the miRNA function in mediating plant drought tolerance. Briefly,
the seedlings at the third leaf stage under normal condition as aforementioned
were cultured in a MS solution containing PEG-6000 (5%, w/v). Three weeks
later, phenotypes and dry mass of the transgenic lines and WT plants were
analyzed. Of these, the plant phenotypes were recorded based on images taken
with a digital camera; the dry mass was derived from the oven-dried samples as
conventional approach. Several photosynthetic traits, including the
photosynthetic rate (Pn), PSII efficiency (ΦPSII) and nonphotochemical
quenching (NPQ), were determined as previous description (Guo et al. 2013).
Assays of
stomata closing rate in transgenic lines
Stomata closing rates (SCR)
representing the extent of plant drought response was evaluated using the
drought-challenged transgenic lines as samples. Briefly, the plants of Sen 2
and WT cultured under normal growth condition were transferred into a modified
MS solution containing 10% PEG (w/v). At times of 0.5 h, 1 h and 2 h under the
treatment, leaf samples of transgenic and WT seedlings at indicated times
together with that prior to treatment (0 h, control) were collected. The
stomata nature of samples after collection was fixed using nail polish oil and
subjected to observation under microscope as described previously (Wei et al. 2020). SCR values in the transgenic and WT
were calculated based on stomata widths at the time points mentioned and those
expressed at 0 h.
Assay of AE
activities and AE family gene expression
Several
cellular reactive oxygen species (ROS) traits, such as the activities of
antioxidant enzymes (AE, superoxide dismutase (SOD), catalase (CAT) and
peroxidase (POD)) and contents of MDA, were measured using the transgenic and
WT plants after drought treatment as materials (Huang et al. 2010). To deepen
understanding of molecular processes underlying the modified AE activities
regulated by TaMIR5086, the genes in AE families (six SOD, six CAT and eleven POD) in T. aestivum were identified
in NCBI GenBank database and were analyzed for transcripts in the
drought-challenged transgenic lines, using qRT-PCR with the specific primer pairs (Table S1). Tatubulin, a wheat constitutive gene, was used for normalization of
the transcripts of the target genes.
Statistics analysis
Gene expression levels, plant dry mass, photosynthetic
traits, stomata closing rate, activities of AE, and contents of MDA in the
experimental materials were determined using four replicates. The Statistical
Analysis System software (SAS Corporation, Cory, NC, USA) was adopted for
analysis of standard errors for averages and significant differences for the
traits among the materials.
Fig. 1: Base pairing
characterization among TaMIR5086 and its target genes
TaTIF (eukaryotic translation
initiation factor s subunit C, TraesCS5B02G410800), TaTP (transport protein section
16, TraesCS6D02G022300), TaRPS
(DNA-RNA directed RNA polymerase subunit beta, TraesCS5D02G383900), TaRPT
(mediator of RNA polymerase II transcription subunit 13, TraesCS5D02G551300), TaSF (sucrose
1-fructorsyltransferease, TraesCS7D02G008700.1), and TaAP (AP-1 complex subunit gamma, TraesCS7D02G128900)
Fig. 2: Expression patterns of TaMIR5086 and the target genes
under drought and recovery conditions
A, TaMIR5086; B, TaTIF and TaTP; C, TaRPS
and TaRPT; D, TaSF and TaAP. 0 h, prior to treatment; 6 h, 12 h, 24
h, and 48 h, times after drought treatment; R6 h, R12 h, R24 h, and R48 h,
times after normal recovery treatment. Data are normalized by internal standard
Tatubulin
and shown by averages plus standard errors
Results
Characterization of the target’s genes of TaMIR5086
In total of six target genes
putatively interacted by TaMIR5086 were identified, including those encoding
eukaryotic translation initiation factor subunit C (TaTIF, TraesCS5B02G410800), transport protein section 16 (TaTP, TraesCS6D02G022300), DNA-RNA
directed RNA polymerase subunit beta (TaRPS,
TraesCS5D02G383900), mediator of RNA polymerase II transcription subunit 13 (TaRPT, TraesCS5D02G551300), sucrose
1-fructorsyltransferease (TaSF,
TraesCS7D02G008700), and AP-1 complex subunit gamma (TaAP, TraesCS7D02G128900). The pairing features at nucleic acid level
among the target genes and TaMIR5086 are shown in Fig. 1. Prediction analysis
on the target genes suggested that they are functional in diverse biological
processes, including transcription (i.e., TaRPS and TaRPT),
translation (TaTIF and TaABP),
protein degradation (TaAP), and trafficking (TaTP and TaSF).
Therefore, TaMIR5086 exerts distinct biological functions in plants by
regulating its target genes.
Expression behaviors of TaMIR5086 and its target genes
TaMIR5086 confers plants improved growth and dry mass production
capacities
OE 2 and OE 3, two lines
overexpressing TaMIR5086, were selected for addressing the role of this miRNA
in regulating plant drought response. Under the normal condition, the
transgenic lines were comparable on phenotypes and dry mass with WT (Fig. 3A–C).
Under drought treatment, OE 2 and OE 3 were much better on the growth features and
biomass than wild type (Fig. 3A–C).
Therefore, TaMIR5086 is an essential regulator in mediating the drought
adaptation of plants.
Photosynthetic parameters and stomata closing rates in transgenic lines
Photosynthetic
parameters and the stomata movement characterization in the transgenic lines
were measured under drought condition. Like growth features and biomass shown in
the transgenic and WT plants, the photosynthetic traits, namely, Pn, ΦPSII and NPQ, were similar each other among
the transgenic lines and WT under normal condition and were modified
drastically under drought treatment. Of which, Pn and ΦPSII were higher and NPQ lower in OE 2 and OE
3 than the WT plants under drought condition (Fig. 4A–C). Assays of the stomata
closing rates (SCR) revealed that the stomata movement in OE 2 and OE 3 was
sensitive in response to drought with respect to wild type; the SCR values were
reduced swiftly in the transgenic lines following a 3 h-regime drought
progression (Fig. 4D). Therefore, TaMIR5086 positively regulates the photosynthetic function and
adjusts the stomata response to drought, by which to impact on the plant drought
acclimation process.
Activities of AE proteins and AE family gene expression
Activities of SOD, CAT, and
POD involving cellular ROS homeostasis together with MDA amounts were assayed
in the experimental materials. Under normal condition, OE 2 and OE 3 displayed
similar behaviors on the SOD, CAT, and POD activities and MDA amounts. Under
drought treatment, the transgenic lines showed higher activities of SOD, CAT,
and POD and lower MDA amounts than the WT plants (Fig. 5A–D). Therefore, the
ROS-associated traits shown in transgenic lines were consistent with the
photosynthetic function, biomass, and growth feature mediated by TaMIR5086.
These results suggested that improvement of cellular ROS homeostasis underlying
miRNA regulation positively modulates plant drought response.
Fig. 3: Phenotypes and biomass of the transgenic lines with TaMIR5086
overexpression under drought treatment
A, phenotypes under
normal condition; B, phenotypes under drought treatment; C,
biomass. WT, wild type; OE 2 and OE 3, two transgenic lines with TaMIR5086
overexpression. In C, data shown are average plus standard error with symbol *
to represent statistically significant compared with WT (P < 0.05)
Fig. 4: Photosynthetic parameters and leaf water loss rates in the transgenic and
wild type plants under drought treatment
A, Pn; B, ѱPSII; C, NPQ; D, leaf water loss
rates. WT, wild type; OE 2 and OE3, two transgenic lines with TaMIR5086
overexpression. Data shown are averages plus standard errors with symbol * to
represent statistically significant compared with WT (P < 0.05)
Fig. 5: Activities of antioxidant enzymes and contents of MDA in
the transgenic and wild type plants under drought treatment
A, activities of SOD; B,
activities of CAT; C, activities of POD; D, contents of MDA. WT,
wild type; OE 2 and OE3, two transgenic lines with TaMIR5086 overexpression.
Data shown are averages plus standard errors with symbol * to represent
statistically significant compared with WT (P
< 0.05)
Fig. 6: Expression patterns of genes encoding antioxidant enzymes
in the transgenic and wild type plants under drought treatment
A, genes of SOD family; B,
genes of CAT family; C, genes of POD family. WT, wild type; OE 2 and
OE3, two transgenic lines with TaMIR5086 overexpression. Data shown are
averages plus standard errors with symbol * to represent statistically
significant compared with WT (P < 0.05).
The expression levels in wild type plants were set as 1
The transcripts of the AE
family genes in the samples were analyzed under drought condition. The SOD gene
TaSOD3, CAT gene TaCAT5 and POD gene TaPOD9 displayed
elevated transcripts in OE 2 and OE 3 compared with WT plants (Fig. 6A–C),
whose modified patterns in expression contrasted with other ones that were
unaltered on transcription in the assayed samples (Fig. 6A–C). These results
indicated that distinct AE family genes, including TaSOD3, TaCAT5
and TaPOD9, are transcriptional response to drought underlying
regulation of TaMIR5086. These differential genes thus contribute to cellular
ROS homeostasis and drought adaptation of plants by regulating the AE
activities.
Discussion
The growth, development, and stress responses of plants
underlying miRNA regulation are dependent on target genes interacted by the
miRNA members, via
posttranscriptional or translational regulation mechanisms (Ferdous et al. 2015; Basso et al. 2019). In this investigation, predicting target genes underlying
TaMIR5086 revealed that this miRNA targets six genes, which are associated with
diverse biological processes. Therefore, TaMIR5086 constitutes the miRNA/target
modules to exert roles in distinct pathways impacting plant growth and stress
responses. Previously, it has reported that the modified growth and stress
responses underlying miRNA regulation are largely dependent on the target genes
that encode transcription factors (Samad et al. 2017). In this study, six target genes interacted by TaMIR5086
are categorized into different functional classes, including transcription,
translation, protein degradation and trafficking. Therefore, TaMIR5086 involves
regulation of plant growth and stress response through modulating diverse
biological processes. Further functional analysis of these target genes can
benefit understanding of the regulation mechanisms of the miRNA members.
The
transcription process of miRNA members is similar to that of mRNA molecules,
which is involved in a suite of molecular processes mediated by various
regulatory factors, such as enzyme Pol II recruit to promoter region mediated by distinct
transcriptional coactivators (Kim et al.
2011; Megraw et al. 2016), the motif
of TATA box situated in promoter location (Xie et al. 2005) and the regulatory
elements located at distinct promoter positions (Hajdarpašić and
Ruggenthaler 2012; Liang et al. 2012). Thus far, the cis-regulatory motif referred to as CRE
(with motif CCGCGT, CACGTGT and AAGTCAA) has been confirmed to be enriched in
gene promoters, playing critical roles in regulating transcription efficiency
of the stress-associated genes, given their interaction with the transcription factors in the
bZIP family (Ma et al. 2012). In this investigation, expression
analysis on TaMIR5086 revealed that it is sensitive in response to drought
stress, with a mode to be temporal-dependent across the drought regime and
recovery transcripts following the normal recovery condition. Therefore,
further investigation of the cis-acting
elements, such as CRE and other ones involving modulation of miRNA
transcription under drought stress, can provide insights in understanding of
the transcription mechanisms of the miRNA members upon drought stressor.
The miRNA members have been
documented in plant tolerance to various abiotic stressors. For example, miR319
member of Arabidopsis increased transcripts upon drought stress. The transgenic
lines with miR319 overexpression improved growth traits and enhanced plant
drought tolerance through enhancing leaf wax accumulation and water holding
ability, given that it downregulates expression of AsPCF5, AsPCF6, AsPCF8 and AsTCP14 that encode
the TCP TF proteins (Nag et al. 2009). In this
investigation, transgene evaluation on TaMIR5086 confirmed its positive roles
in regulating plant drought acclimation; the lines overexpressing the miRNA
improved plant phenotypes, biomass, and photosynthetic traits under drought
condition. These findings indicated that TaMIR5086 acts as one of molecular
indices for evaluating drought tolerance across the wheat varieties, which acts
as a valuable target for molecular breeding of the drought-tolerant cultivars
in T. aestivum.
Stomata movement is one of the important acclimation mechanisms for
plants to cope with drought stress (Kollist et
al. 2014; He et al. 2018).
Investigations have indicated that stomata closure is mediated byenhanced
abscisic acid (ABA) levels due to promoted biosynthesis and reduced degradation
of ABA under drought (Boursiac et al.
2013; Song et al. 2014; He et al. 2018). Thus far, the ABA signaling
pathways associated with stomata closing upon drought signaling, such as
characterizations of the ABA receptor members, namely RCAR, PYR1 and PYL,
interaction mechanisms of ABA receptors with type 2C protein phosphatases
(PP2C), activation of the SNF1-related protein kinase OPEN STOMATA1
(OST1)/SnRK2, have been extensively investigated in the model plants (Park et al. 2009; Tan et al. 2018). In this study, analysis of the SCR behavior in lines with TaMIR5086
overexpression revealed its promoted stomata closing nature upon drought, which
suggested that the modified stomata movement acts as one of the acclimation
pathways for the miRNA-mediated drought response. Therefore, further
investigation of the TaMIR5086-mediated ABA signaling pathway can help
understanding of the molecular mechanisms of plant drought response underlying
miRNA regulation.
Reactive oxygen species (ROS)
have been documented to be over-accumulated in plants upon drought conditions,
which negatively impact on plant drought tolerance due to deterioration of
structures of protein, lipid, and nucleic acid and decrease of cell vigor (Gill
and Tuteja 2010; Hasanuzzaman et al.
2020). The antioxidant enzymes (AE), including superoxide dismutase, catalase,
and peroxidase, positively impact on plant adaptation to diverse stresses
through improvement of cellular ROS homeostasis (You and Chan 2015; Lin et al. 2019). For example, the alfalfa
lines overexpressing a MnSOD gene of N. plumbaginifolia endowed plants improved growth and yield
under drought condition (McKersie et al.
1996). Likewise, overexpression of a cytosolic SuZnSOD gene of A. marina
led to enhanced drought tolerance in rice plants (Prashanth et al. 2008). In this investigation, the
transgenic lines overexpressing TaMIR5086 elevated the AE activities (i.e.,
SOD, CAT and POD ones) and reduced the contents of MDA under drought compared
with WT, suggested that the AE enzymes improve plant drought response
underlying miRNA regulation, via
modulating the cellular ROS homeostasis. Moreover, expression analysis revealed
that the SOD gene TaSOD3, CAT gene TaCAT5 and POD gene TaPOD9 upregulated expression levels in transgenic lines under
drought stress. These findings suggested that the contribution of distinct genes
in AE families to the improved antioxidant enzyme functions. Thus far, the
internal relations between ROS and stomata movement under drought stress have
been recorded. For example, the H2O2
production underlying ABA signaling pathway initiated by drought is promoted by plasma membrane-bound NADPH oxidase (RBOH), which
promotes the hydrogen peroxide biosynthesis (Sirichandra et al. 2009; Czékus et al.
2020), by which to activate the calcium channels that elevate Ca2+
level in guard cells, promoting depolarization of membrane and the closure of
stomata (Song and Matsuoka 2009). In this investigation, the modified stomata
closing rate and ROS homeostasis under drought were shown to be regulated
underlying TaMIR5086. It will be valuable to further elucidate the regulators
to modulate the stomata movement and ROS homeostasis in the TaMIR5086
transgenic lines, which benefit to deepen understanding of the plant drought
response mediated by distinct miRNA members.
Conclusion
TaMIR5086 has six target genes that are
involved in diverse biological processes. The transcripts of TaMIR5086 were
upregulated whereas its target genes downregulated upon drought stress, which
suggested the regulation of the target genes underlying miRNA via posttranscriptional mechanism. TaMIR5086
conferred plants improved drought tolerance, being ascribed largely to
improvement of stomata movement, ROS homeostasis and photosynthetic function.
The AE genes TaSOD3, TaCAT5, and TaPOD9 upregulate expression in the drought-challenged lines overexpressing
TaMIR5086. Elevation of stomata closing and antioxidant enzyme functions
contributes to plant drought adaptation and TaMIR5086 enhances plant drought
tolerance and is useful for generating wheat cultivars sharing drought
tolerance nature. Our findings provide insight into plant drought adaptation
underlying miRNA members and elucidate valuable target for generating crop
germplasms to be drought tolerant.
Acknowledgments
This work was supported by the National
Natural Science Foundation of China (No. 31872869) and College Student
Innovation and Entrepreneurship Training Program Project (s202010086047).
Author Contributions
YZ,
MZ, GS, LW, ZW, YZ, and HX planned the experiments, KX interpreted the results and
made the write up, CN statistically analyzed the data and made illustrations.
Conflict of Interest
The
authors declare there is no conflict of interest.
Data Availability
Not applicable.
Ethics Approval
This
study is in accordance with the ethical standards.
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