Plant Molecular Biology

https://doi.org/10.1007/s11103-023-01389-7

Genetic diversity, transcript heterogeneity and allele mining

of TaSKP1‑6B‑4 gene variants across diverse genotypes under terminal
heat stress and genome wide characterization of TaSKP1 gene family
from bread wheat (Triticum aestivum L.)
Praful Jaiswal1,2 · Akshay Singh3 · Kriti Bajpai4 · Kabitha Tripathi1 · Anant Narayan Sahi2 · Sharmistha Barthakur1

Received: 25 March 2023 / Accepted: 9 October 2023

© The Author(s), under exclusive licence to Springer Nature B.V. 2023

Abstract
SKP1 (S-phase kinase protein1) is an essential regulatory component of SCF (Skp1-cullin-F-box) E3 ubiquitin ligases involved in
maintenance of cellular protein homeostasis through ubiquitin mediated proteasome system (UPS). UPS play a key role in stress
response and grain yield. Earlier, we isolated TaSKP1-6B-4, highly induced in flag leaf tissues (Accession No. KJ830759.1) of devel-
oping wheat caryopses under heat stress. To further assess the functional role of SKP1, genetic variability analysis was carried out in
a panel of 25 contrasting germplasm through extensive phenotyping and transcript profiling of TaSKP1-6B-4 during anthesis under
ambient and terminal heat stress (THS) in field experiments for two consecutive years. The analysis of variance revealed significant
­ 2(%), GCV, PCV, GA and GA% mean observed in tiller number per plant (23.81, 17.65,
variations for all the traits studied. Higher H
5.71, 28, 30.86%) and grain number per head (30.27, 82.79, 60.16, 105.00, 108.64%) under THS over ambient temperature. Higher
fold induction of TaSKP1-6B-4 transcripts was recorded in 10 genotypes viz. HD2967 (9.9), IC145456 (6.18) in flag leaf; while
C-306 (15.88), RAJ3765 (8.37) in ear head. Allele mining of SKP1-6B-4 showed genotypic sequence variations. Whole genome
wide search of SKP1 gene family identified 95 SKP1 genes which were structurally characterized. Grain yield, leaf senescence and
other agronomic-morpho-physiological parameters combined with transcript profiling, cvHD2967, was found to be the best posi-
tively responsive to THS which by pedigree was not heat tolerant. We report a novel 2 year comprehensive field based analysis on
collective genetic variability and SKP1/UPS modulation under a natural environmental setting. The data reveals potential functional
role of UPS under THS and tolerant cultivars can be further utilized for clarifying the role of UPS mechanistically at the molecular
level and for developing terminal heat stress tolerant wheat.

Key messages
Genetic diversity and transcript profiling of heat inducible gene in a field experiment collectively identify terminal heat stress
tolerant wheat cultivars and involvement of UPS to tackle climate adversity.

Keywords Terminal heat stress · Anthesis stage · Heat tolerant · Flag leaf · Ear head · SKP1

1
* Sharmistha Barthakur ICAR-National Institute for Plant Biotechnology,
Sharmistha.Barthakur@icar.gov.in New Delhi 110012, India
2
Praful Jaiswal Amity Institute of Biotechnology, Amity University, Noida,
jaiswalpraful1987@gmail.com U.P, India
3
Akshay Singh ICAR-National Bureau of Plant Genetic Resources,
akshaybioinfo@gmail.com New Delhi 110012, India
4
Kriti Bajpai ICAR- Indian Agriculture Research Institute,
kritibajpai123@gmail.com New Delhi 110012, India
Kabitha Tripathi
bioinfokabitatripathy@gmail.com
Anant Narayan Sahi
ansahi@amity.edu

13
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Abbreviations
TN/P Tiller number/plant
FLL Flag leaf length
FLW Flag leaf width
PH Plant height
PDL Peduncle length
SN/P Spike number/plant
SL Spike length
GN/H Grain number/head
TW Test weight
DOB Days of booting
DOA Days of anthesis
DOPA Days of post anthesis
H2 (%) Analysis of heritability (percent)
GCV Genotypic coefficient of variation
PCV Phenotypic coefficient of variation
GA Genetic advance
GA% mean Genetic advance value % mean
HS Heat stress (HS)
ROS Reactive oxygen species
Introduction
Wheat (Triticum aestivum L.) is a universal and important
food crop, and improving its resistance to stresses is an
important research challenge around the world. The effects
of climate change have weakened progress in global wheat
yields (Lobell and Field 2007; Zhao et al. 2017; Ullah et al.
2019) and limited global wheat production (Asseng et al.
2015; Chatzopoulos et al. 2021). The forecast for world
wheat production in 2023 has been scaled back moderately
since the previous year, but standing at 782.7 million tons;
however, FAO still predicts global wheat production to grow
this year. In India wheat production was 106 million metric
tons, down 1 percent from last year, due to unusual early
hot weather, nevertheless up by 4 percent from the 5-year
average. During the reproductive stage, an increase in tem-
perature of even 1 °C will cause grain yields to decrease
(Bennett et al. 2012; Mirosavljević et al. 2021). According
to the IPCC report in 2014, observations show that atmos-
pheric temperatures have increased since the turn of the
twentieth century, and by the year 2050 they are expected
to rise by another 1.0–1.7 °C. Consequently, global climate
changes will cause wheat to experience greater levels of heat
stress (Liu et al. 2017). In most cases, plants have an inbuilt
defense system that mitigates these stresses, but only up to a
certain level of tolerance. The natural defense system is dis-
rupted under severe stress conditions, which leads to injury
and abnormal physiological conditions within the cell, ulti-
mately leading to yield losses (Ma et al. 2018).
In wheat, heat stress affects every developmental stage,
but it is more pronounced at the reproductive stage (Lobell
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Plant Molecular Biology
et al. 2012; Frieler et al. 2017; Tricker et al. 2018; Zampieri
et al. 2018). Heat stress influences seed germination, grain
filling duration, grain number, Rubisco enzyme activity,
photosynthetic capacity, pigmentation, accelerated senes-
cence, chlorophyll content, phasic development, and respi-
ration, which increase evaporation in kernels. These actions
affect wheat yield by reducing yield potential. Heat stress
(HS) produces reactive oxygen species (ROS), leading to
damage of nucleic acids, protein oxidation, and lipid peroxi-
dation (Narayanan et al. 2016; Djanaguiraman et al. 2018).
The rise in daily average temperature, up to 30 °C, increases
dough strength, while temperature above this threshold value
(35 °C to 40 °C), even for periods of only few days disrupts
tapetal cells during pollen development, causing collapsed
and desiccated pollen grains with irregular surface patterns
in wheat as well as decreasing dough strength (Randall and
Moss 1990; Bokshi et al. 2021).
Plants being sessile can dramatically alter their gene
expressions in response to various stress signals through a
set of morphological, physiological, and molecular changes
that can harm the stability of plant growth and productivity
(Castelán-Muñoz et al. 2019). Plants use numerous mecha-
nisms, which involve intricate but well-regulated signaling
pathways, to deal with environmental stress. Plants can be
effectively regulated by controlling the gene expression in
response to heat stress. In addition to transcription factors
and functional proteins, other pathways and their integral
component genes play a role in plant heat tolerance. One
such system is the ubiquitin proteasome system (UPS) which
regulates the abundance of numerous enzymes, structural
and regulatory proteins ensuring proper cellular function
(Miricescu et al. 2018; Stone 2019). Plants utilize the UPS
to facilitate cellular changes required to respond to and tol-
erate adverse growth conditions. The UPS is involved in
a broad array of cellular processes, such as signaling, cell
cycle, vesicle trafficking, and immunity (Vierstra 2012;
Mackinnon and Stone 2022). In this system ATP-dependent
reaction cascades, polymeric ubiquitin chains recognize and
modify intracellular proteins. Selective protein degradation
by the UPS proceeds from the ligation of one or more ubiq-
uitin proteins to the α-amino group of a lysine residue within
specific target proteins catalyzed by E1 ubiquitin-activating
enzymes, the E2 ubiquitin-conjugating enzymes, and the
E3 ubiquitin ligase enzymes. Ubiquitination can regulate
cellular signaling processes vide mono-ubiquitination or
multi-ubiquitination associated with endocytosis, protein
sorting, gene expression, and various other cellular pathways
(Mukhopadhyay and Riezman 2007; Adams and Spoel 2018;
Stone 2019; Xiong et al. 2013).
SKP1 plays a crucial role in connecting CUL1 and the
F-box protein of the SCF-type E3 ubiquitin ligases, which
are the most extensively studied type of ubiquitin ligase.
SKP1 is a protein of approximately 160 amino acids that
Plant Molecular Biology
plays an important role in cell-cycle development as well as
in vegetative and flower development (Jackson et al.2000;
Metzger et al. 2012, 2014). Different SKP1 genes appear
in plants as a result of duplication events leading to diverse
functions (Kong et al. 2004, 2007). ASK1 and OSK1 genes
from Arabidopsis and rice had been studied in context to
gene evolution (Kong et al. 2004, 2007). Compared to other
members of the SCF complex, SKP1-like proteins in plants
have been less studied. SKP1 proteins also have the potential
to determine the specificity of SCF complexes. Despite the
importance of the SCF complex, there have been few reports
of systematic surveys of interactions between SKP1-like pro-
tein as well as other components of SCF complex. Except
for our work on terminal heat stress induction of SKP1, we
did not come across any other reports of SKP1 modulation
under high temperature stress (Jaiswal et al.2022).
Earlier we isolated the coding region of TaSKP1-6B-4
(TraesCS6B02G0043004) after comprehensive transcription
expression profiling. The gene is highly induced in differ-
ent tissues of developing caryopses of wheat when exposed
to high temperature stress at the anthesis stage (Accession
numberKJ830759).
To further elucidate the role of UPS in imparting heat
stress tolerance during the most vulnerable stage of the crop
at anthesis; we hypothesized that a combined genetic vari-
ability analysis using a panel of wide ranging differentially
heat responsive wheat germplasm and transcript profiling of
a UPS critical gene can help identify THS tolerant genotypes
for further mechanistic analysis. Thus this work was initiated
with the following objectives: (i) to expose diverse germ-
plasm to high ambient temperature in situ, and carry out
phenotyping for two consecutive years in natura, (ii) Tran-
script profiling of an UPS component TaSKP1-6B-4highly
induced by terminal heat stress, isolate alleles from thermo
tolerant genotypes and whole genome analysis of TaSKP1
gene family along with their structural characterization to
understand the genomic architecture.
Allele mining of CDS of 10 full-length variants of
TaSKP1-6B-4 from 10 diverse genotypes was carried out
from developing spikes after exposing to high temperature
and transcript profiling under ambient and high tempera-
ture using qRTPCR. Genome wide analysis of TaSKP1 like
genes was done with retrieved data from the latest release of
the wheat genome database of International Wheat Genome
Sequencing Consortium (IWGSC) and 95 SKP1 genes were
identified (RefSeq v2.1: https://​urgi.​versa​illes.​inrae.​fr). A
comprehensive and systemic analysis of this SKP1 gene
family, protein structures, cis acting elements, phylogenetic
relationships, and chromosome distribution was carried out.
Our results demonstrate that a field based analysis of tran-
script heterogeneity of a positively modulated heat respon-
sive regulatory gene and genetic variability analysis can col-
lectively identify grain filling stage tolerant germplasm. To
the best of our knowledge this is one of the first field based
2 year study evaluating the role of UPS in terminal heat
stress tolerance of bread wheat. This is a realistic simulation
of climate induced high temperature stress relative to fixed
temperature laboratory study.
Materials and methods
Selection of genotypes
Wheat genotypes used in this study were collected from
ICAR-NBPGR, New Delhi, India. The experiment was con-
ducted in two consecutive years 2016–2017 and 2017–2018
in the wheat growing Rabi season of India. A set of 25
diverse wheat genotypes were selected for use on the basis
of previous documented reports of differential tolerability
to heat stress (Table S1a) (Dutta et al. 2015; Kumar et al.
2016). We also included in this miscellaneous group of gen-
otypes some newly identified heat tolerant germplasm which
were not yet documented to re confirm their heat respon-
siveness. In the second year 2017–2018, 14 genotypes were
screened out on the basis of phenotypic analyses carried out
during terminal heat stress applied in the first year. At the
anthesis stage, this group of wheat genotypes (Table S1 b) in
the second year was exposed to heat stress again to evaluate
the transgenerational heat stress response.
Experimental design and environmental growth
conditions
The experiment was conducted at the Indian Agricultural
Research Institute (IARI), New Delhi, India. It has semi-
arid, subtropical climate and surface soil (0–30 cm) of fine
loamy, mixed, slightly alkaline, moderately permeable
belonging to the hyper thermic family of the typichaplus-
tept (old alluvium) soil which was used to fill the pots. In the
entire experiment, a completely randomized design (CRD)
was used with three replications per genotype. The seeds
were sown in plastic pots, size 20 cm diameter and 27 cm
in height and each pot contained 5 kg of soil that was mixed
with 500 g of vermicompost obtained from the Biomass unit,
ICAR-IARI, New Delhi, India. Ten seeds were planted and
extra plants were thinned out one week after the emergence
of plants to retain 5 healthy plants per pot. These pots were
well irrigated and during sowing a first dose of 20 ml of
soluble fertilizer (N:P:K ratio of 12:6:3; IFFCO, India) was
applied to each pot and a second dose was provided after
the anthesis stage. Cultural practices were carried out as per
standard procedures. At the 14th day of anthesis (Zadoks
growth stage 60 i.e. the beginning of pollination) plants
were exposed to heat stress under a heat trap chamber in
in situ condition. The plants were divided into two groups,
13

approaching anthesis, half of the plants were exposed to
heat stress under heat trap chamber and the other group was
kept in ambient conditions as heat stress control. The plants
were categorized on the basis of days taken to enter in to
the anthesis stage and thereby maturity into two groups.
This was because the germplasm group was heterogeneous
and did not have uniform and variable entry or transition
into reproductive or anthesis growth stage. Heat treatment
was performed on the first set of plants from February 23rd
to 25th, and on the other set of plants from March 23rd to
25th, 2017 for 3 consecutive days at peak heat during the
afternoon from 11:00 am to 03:00 pm for 4 h. During the
second year, 2018, the same heat treatment methodology
was utilized from March 12–14 (Supplementary file 1).
The heat trap chamber, which is made up of aluminum and
covered with transparent plastic sheets with dimensions
front 64 × 58 and side 62 × 79 inches and a roof top open air
window (1 × 1ft) to maintain ventilation keeping the same
concentrations of carbon dioxide and air humidity on the
inside and outside and light transmittance of 92% from out-
side (Fig. 1). The chamber maintained a temperature 4 to
5 °C above the existing ambient temperature (Khomdram
and Barthakur 2015). The ambient temperature as well as the
heat trap chamber temperature were measured hourly dur-
ing treatments. After the treatment tissue samples were col-
lected from flag leaves and ear heads under both conditions,
frozen in liquid nitrogen and stored at − 80 °C for further
RNA isolation and transcript profiling as well as for morpho-
physiological observations. At the end of the experiment
separate spikes were collected for grain evaluation data and
further raising of plants in the next season.
Evaluation of morpho‑physiological and yield
related parameters
After the heat exposure of the plants, 12 morpho-physio-
logical traits including as Tiller number/plant (TN/P), Flag
leaf length (FLL), Flag leaf width (FLW), Plant height (PH),
Peduncle length (PDL) along with Chlorophyll content
which was recorded in the beginning days of booting (DOB),
days of anthesis (DOA) and days of post anthesis (DOPA).
The development stages were monitored of the tagged
plants; DOB (ZGS 47, 1st spikelet of spike just visible),
DOA (ZGS 50, 50–80% of spikes were visible and anther
color was green) and DOPA (ZGS 59, anther color was off
white or fallen) (Zadoks et al. 1974). In continuation to this,
the number of productive tillers TN/P was recorded in main
stem as well as sub stem; the completely expanded FL data
were collected for flag leaf length, FLW measurement from
widest part of the flag leaf, PL from last node of flag leaf to
leaf collar and PH from soil surface to spikelet of the plants.
Chlorophyll content was measured on the main stem of flag
leaves of five tagged plants per pot, using a self-calibrating
13
Plant Molecular Biology
SPAD chlorophyll meter (Model 502, Konica Minolta) and
values were expressed in SPAD units (Reynolds et al. 1998).
At maturity, individual spikelet or ear heads were harvested
and grains collected during both the years. Yield component
data were estimated as number/plant (SN/P), spike length
(SL), Grain number/head (GN/H), thousand grain weight
(TW) to determine grain yields. Three pots for each treat-
ment were taken as three biological replicates.
RNA isolation, cDNA synthesis and transcript
expression profiling
Total RNA was isolated from flag leaves and ear head using
Trizol (Biobasic, Canada) fallowed by DNaseI treatments
following the manufacturer's instructions (Promega). DNaseI
treated RNA was reverse transcribed with a cDNA synthe-
sis kit and synthesized from 3 μg of total RNA using the
ImpromII reverse transcriptase (Promega, USA).The inter-
nal check with 18SrRNA gene (Accession no. Y357916)
primers and expression analysis was done with gene-specific
primers for amplification (Table 1). Primer sequences were
designed from the available SKP1 sequences cloned earlier
in the lab (Accession number KJ830759) and from available
public databases at NCBI (www.​ncbi.​nlm.​nih.​gov.​in) (Sup-
plymentary file 3). PCR reactions were performed in 20 μl
volume of reaction mixture containing 1X green master mix
(Takara Bio Inc, Shiga, Japan) and 10 nM of each forward
and reverse primer. The whole reaction was incubated in a
thermal cycler program of initial denaturation at 94 °C for
4 min followed by 35 cycles of 94 °C for 45 s-denaturation,
60 °C for 45 s-annealing, 72 °C for 1 min-extension and a
final extension at 72 °C for 10 min (Biometra, Gottingen,
Germany) for both the primers 18SrRNA and TaSKP1-
6B-4expression profiling.
Quantitative reverse transcription PCR (qRT‑PCR)
analysis
The mixed panel of fourteen genotypes screened out from
the first year results were used in the second year to tran-
script profile gene expression using qRT PCR. Samples
from flag leaf and ear head tissues collected from plants
with three replications for each sample. The real-time
quantification was performed in Real Time PCR System
(Eppendorf) and analyzed with the software version 2.0.4.
The reaction mixture (10 µl) contained 0.5 µl of cDNA
sample (equivalent to 10 ng of the initial total RNA),
0.4 µM of each primer, and 5 µl of universal SYBR Green
q-PCR Superscript master mix (Agilent) and 0.2 µl of Rox
reference dye. For a negative control reaction, no template
was added to the reaction mixture, which resulted in no
detectable fluorescence signal from the reaction. qRT-PCR
conditions were set as follows: initial denaturation for 30 s
Plant Molecular Biology
Fig. 1  Daily temperature data recorded during in situ heat exposure
period from 11.00 am to 03.00 pm for 3 consecutive days during the
growth period in 2 years 2016–2017 and 2017–2018. b A picture of
at 95 °C, followed by 40 cycles of denaturation at 95 °C
for 5 s, and annealing and extension for 34 s at 60 °C.
After amplification cycles, each reaction was subjected to
melting-temperature analysis to confirm single amplified
products: 95 °C for 15 s, 60 °C for 60 s, 1% slope eleva-
tion until 95 °C and held for 30 s, followed by final step
at 60 °C for 15 s.
the heat trap chamber used in the experiments for heat stress incuba-
tion. c In situ heat exposure under heat trap chamber
Full length TaSKP1‑6B‑4 CDS isolation from different
wheat genotypes
Ten most promising wheat genotypes from the second
year analyses were selected for isolation of full length cod-
ing region of TaSKP1-6B-4 alleles. The cDNA fragments
were, amplified by PCR with appropriate primer sequence
(Table 1) using emerald green master mix (Takara Clon-
tech). Amplified products were excised from 1% agarose gel
and purified by the gel extraction kit (Promega). The ampli-
cons were cloned into the pGEMT easy vector (Promega)
13
 Plant Molecular Biology

Table 1  Details of primers used in the present study

Primer Name Strand Sequence (5′ > 3′) Length (bp) Tm (°C) Purpose of use

18S rRNA F 5′ TTT​GAC​TCA​ACA​CGG​GGA​AA 3′ 180 60 Internal constitutive control

R 5′ CAG​ACA​AAT​CGC​TCC​ACC​AA 3′
TaFSKP1 F 5′ GAG​CGA​TGG​CGG​CCG​CGG​GAGAC 3′ 520 60 cDNA amplification and full CDS cloning
R 5′ ACC​ACT​GAC​CGA​AGA​TGT​TCA​CCA​ 3′
TaSKP1 exp F 5′ TGG​CTG​CCA​ACT​ACC​TGA​ACA 3′ 350 60 cDNA amplification and relative expression
R 5′ ACA​TGT​TCA​CCG​ACA​CCA​CCT 3′
TaSKP1 RT F 5′ TGC​CAA​CTA​CCT​GAA​CAT​CAA 3′ 180 55 Relative expression
R 5′ GTT​GAA​GGT​CTT​GCG​GAT​CT 3′

F the forward primer, R indicates the reverse primer

Table 2  List of wheat genotypes used for isolation of SKP1 alleles the Pfam database and used as a query in the HMMER
accession numbers and CDS length of respective SKP allele search with an E-value cut-off < 0.1. The results were col-
S.no. Wheat genotypes Accession no. CDS lectively used for the presence of SKP1 and SKP1_POZ
length domain using ScanProsite (http://​p rosi​t e.​expasy.​o rg/​
(bp) scanp​rosite/) and the NCBI-Conserved Domain Database
1 HD3090 OK616157 522 (Marchler-Bauer et al. 2017).
2 C-306 OK616158 366
3 IC145456 OK616159 525
4 WR544 OK616160 525 Sequence analysis, chromosomal distribution
5 HD3086 OK616161 525 and phylogenetic analysis
6 RAJ3765 OK616162 525
7 HD2967 OK616163 525 The composition of deduced TaSKP1 proteins with
8 HD2932 OK616164 525 physical and chemical characteristics was analysed with
9 HD2864 OK616165 525 the ExpasyProt-Param tool (http://​web.​expasy.​org/​protp​
10 DL788-2 OK616166 525 aram/). To determine the distribution of the TaSKP1 genes
on the chromosomes of the wheat genome, the GFF3 anno-
tation file was parsed to extract the genomic locations of
through TA cloning and confirmed by colony PCR. Size the predicted TaSKP1 genes. The URGI database of wheat
of the inserts was confirmed by digestion with EcoRI or (https:// ​ w heat- ​ u rgi. ​ versa ​ i lles. ​ i nra. ​ f r/ ​ S eq- ​ R epos​i tory/​
NotI and separated on 1.2% agarose gel. The clones were BLAST) was used to find the total TaSKP1 gene sequences
further sequenced by Senger sequencing (AgriGenome, in whole genome of wheat. The genomic coordinates of
Cochin, India) (Table 2). The sequence homology of all the TaSKP1 genes were used to visualize the detailed location
ten accessions was carried out with various tools available at of each TaSKP1 gene on the wheat chromosome using
NCBI (www.n​ cbi.n​ lm.n​ ih.g​ ov) site, ORF finder searches for Map Chart. In order to study the phylogenetic relation-
open reading frames (ORFs) in the DNA sequence (www.​ ship and evolution of wheat SKP1 like genes, the obtained
ncbi.​nlm.​nih.​gov/​orffi​nder/), coding nucleotide sequence TaSKP1amino acid sequences were compared with the
confirmed with CDD search (www.​ncbi.​nlm.​nih.​gov/​Struc​ orthologues of model monocots Zea mays (Zm), Sorghum
ture/​cdd/​wrpsb.​cgi), for conserved domains and verified bicolour (Sb), Panicum virgatum (Pv), Setaria italica (Si),
sequences were deposited in the gene bank through bankit Oryza sativa (Os), Leersia perrieri (Lp), Brachypodium
tool (submit.ncbi.nlm.nih.gov/about/bankit/). distachyon (Bd), Hordeum vulgare (Hv), Aegilops tauschii
(Aet), and Triticum urartu (Tu).The phylogenetic analysis
Identification of SKP1 like gene family members was carried in Mega-X (www.​megas​oftwa​re.​net) software
from bread wheat tools for finding the evolutionary relationship with other
species of gramene family with default parameters and the
The SKP1 like genes were searched from wheat (Triticum phylogenetic tree was drawn based on the neighbor-joining
aestivum) reference genome v2.1 using the HMMER pro- method with bootstrap value set to 1000 repetitions.
gram version 3.1b2 (http://​hmmer.​janel​ia.​org). The HMM
(Hidden Markov Model) profiles of SKP1 (PF01466) and
SKP1_POZ (PF03931) domains were downloaded from

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Plant Molecular Biology
Gene structure, motif conservation, and regulatory
element analysis
Exon–intron organisation of TaSKP1 genes was performed
by comparing CDS sequences with their respective genomic
sequences using the Gene Structure Display Server (Hu
et al. 2014). Conserved motif sequences across the pre-
dicted TaSKP1genes were analysed using the MEME Suite
v.5.0.2 (Bailey et al. 2009). The search criteria taken for the
identification of motifs by the MEME include a maximum
of 20 motifs to report, and optimal motif width between 8
to 50 residues. Then the conserved motifs were annotated
using InterProScan Search (http://​www.​ebi.​ac.​uk/​Tools/​pfa/​
iprsc​an/). The 2 Kb upstream regions of all TaSKP1 genes
were fetched by a custom Perl script and analyzed to deter-
mine the presence of cis-regulatory elements by the Plant
Cis-acting Regulatory DNA Elements PlantCare database
(Lescot 2002). Then predicted cis-regulatory motifs were
used to draw using TBTools (https://​github.​com/​CJ-​Chen/​
TBtoo​ls/​relea​ses).
Homology modelling and structure analysis
Three dimensional (3D) protein structure predictions of
TaSKP1 proteins were done using the SWISS-MODEL
online server, accessible via the Expasy web server (http://​
web.​expasy.​org). Selection of the best model was made on
the basis of the highest confidence score. The predicted 3D
model was subjected to backbone confirmation evaluation
by Ramachandran plot using Procheck, a unit of the PDB-
sum server (Laskowskiet al.2017) and further confirmed by
RAMPAGE server (Lovell et al. 2003). The final model and
the template were subjected to superimposition for a struc-
tural comparison using the STRAP interface (http://​www.​
bioin​forma​tics.​org/​strap/).
Data analysis
All the statistical analysis of variance (ANOVA) was per-
formed for each character measured in this study. Combined
analysis of variance was performed following the attestation
of homogeneity of variances. In order to calculate Pearson's
correlation coefficients (r), we used the statistical R package
for heat stress and control treatments. By using analysis of
variance, genotypic and phenotypic variances were deter-
mined. A percentage of heritability was calculated based on
the definition provided by Lush (1949) and the calculation
provided by (Hanson 1956). In addition to estimating genetic
advance, a percentage of mean genetic gain was estimated
based on the selection of 5% superior individuals.
Results
During the first year of experiments in 2017, based on the
statistical analysis (ANOVA) of twelve quantitative traits,
twenty five genotypes were analyzed (Table 3) under ambi-
ent and heat stress conditions. The combined analysis of
variance showed significant effect of the source of varia-
tion for all above mentioned traits, indicating that the heat
stress inhibits all the morphological and physiological
traits. On the basis of minimum and maximum range for
selecting the best genotype positively modulated under heat
stress, 14 genotypes were selected for analysis in the next
year (Table 4). Grain yield data for 2017 viz. 1000 grain
weight (TW) increased from 69.20 g in ambient conditions
to 73.70 g under heat stress condition, while in the next
consecutive year i.e., 2018, no significant changes were
observed in the values. On the other hand, plant chloro-
phyll content (SPAD) was determined as 54.87, 58.37and
57.37 under ambient condition whereas 57.03, 56.70 and
58.47under heat stress respectively which showed a mar-
ginal increase under heat stress condition. During 2018 these
values were observed in same normal reduction pattern as
the previous year with values of 49.00, 54.63 and 48.50 in
heat stress condition with comparison to the values of 47.20,
54.73 and 49.50 in ambient condition which were recorded
at the booting, anthesis and post anthesis stages respectively.
In the 2017, minimum range was show by culti-
varsHD3059, EC445278,DL788-2, EC576317 and RAJ3765
for TN/P, FLL, PH, SL and chlorophyll content at DOB,
DOA and DOPA traits respectively (Table 4a).Whereas
the maximum range was exhibited byIC539602, DL788-2,
HUW468, IC539531 for these traits in the FLL, PH, SN/P,
GN/H, under ambient and heat stress conditions respec-
tively. On the basis of these statistical analyses we selected
14 genotypes for further study in 2018. These selected14
cultivars were observed for certain selected traits; C-306,
DL788-2, HD2967 and IC145456 for the traits of FLL, PH,
PL and SL showing minimum range while maximum range
was shown by HD3086 for FLL, FLW, DOA and DOPA.
After further phenotypic data analysis we have found that
RAJ3765 showed maximum SL as well as highest grain
number per head. The cultivar HD2967 showed maximum
chlorophyll content in the DOB stage under heat stress con-
ditions (Table 4b). The results indicate that the genotypes
have different adaptive characteristics under different envi-
ronmental conditions with various genotypes exhibiting dif-
ferent levels of heat tolerance here. In order to determine
which genotype’s are heat-tolerant, these tolerance indices
can be used. These indicators can measure a genotypes' abil-
ity to maintain their productivity under stress conditions.
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 Plant Molecular Biology

Table 3  Analysis of variance (ANOVA) of twenty five (25) and fourteen (14) genotypes in 2017 and 2018, respectively years with twelve (12)
traits under ambient and heat stress condition were analysed in Rabi season both the years, respectively
(a) 2017
Traits TN/P FLL FLW PH PDL SN/P SL GN/H TW DOB DOA DOPA

Ambient
Min 2.67 16.17 1.63 74.37 18.90 1.33 7.63 26.67 20.09 38.10 38.93 36.63
Max 7.00 22.97 2.73 128.10 36.17 3.67 15.87 59.33 69.20 54.87 58.37 57.37
Mean 4.99 19.86 2.23 88.39 30.40 2.46 13.04 39.30 38.87 45.89 47.19 47.93
C.D 1.79 1.38 0.21 3.50 2.14 1.00 1.81 10.87 12.60 3.64 4.43 3.48
SE(m) 0.63 0.48 0.08 1.23 0.75 0.35 0.63 3.81 4.41 1.27 1.55 1.22
SE(d) 0.88 0.69 0.11 1.74 1.06 0.50 0.90 5.38 6.24 1.80 2.19 1.72
C.V 21.71 4.22 5.79 2.41 4.27 24.67 8.42 16.78 19.66 4.81 5.69 4.40
Heat stress
Min 3.00 15.23 1.67 76.43 19.80 1.33 8.45 16.89 23.80 36.43 37.83 36.57
Max 8.00 22.97 2.73 131.60 35.53 3.33 15.53 78.67 73.70 57.03 56.70 58.47
Mean 4.85 19.93 2.21 88.50 30.43 2.32 13.06 38.59 41.38 45.88 47.60 47.57
C.D 1.71 1.04 0.21 3.70 2.20 N/A 1.63 13.92 15.15 3.64 3.45 2.99
SE(m) 0.60 0.36 0.07 1.30 0.77 0.46 0.57 4.87 5.31 1.27 1.21 1.05
SE(d) 0.85 0.52 0.11 1.83 1.09 0.65 0.81 6.89 7.50 1.80 1.71 1.48
C.V 21.36 3.16 5.79 2.54 4.39 34.56 7.55 21.87 22.21 4.81 4.40 3.82
(b) 2018
Traits TN/P FLL FLW PH PDL SN/P SL GN/H TW DOB DOA DOPA

Ambient
Min 3.33 10.10 1.07 65.00 13.77 2.00 8.94 18.33 21.49 35.17 25.50 25.10
Max 6.00 19.17 1.83 104.50 30.37 4.33 14.70 40.50 48.81 47.20 54.73 49.50
Mean 4.86 14.69 1.34 80.86 19.88 2.90 12.55 30.86 36.52 41.04 36.30 37.07
C.D 1.10 1.38 0.15 2.62 1.41 1.01 1.37 8.57 7.23 2.93 2.97 3.42
SE(m) 0.38 0.47 0.05 0.90 0.48 0.35 0.47 2.93 2.47 1.00 1.02 1.17
SE(d) 0.53 0.67 0.07 1.27 0.68 0.49 0.66 4.15 3.50 1.42 1.44 1.66
C.V 13.42 5.57 6.78 1.92 4.21 20.57 6.48 16.46 11.73 4.23 4.84 5.47
Heat stress
Min 3.33 10.60 1.07 64.47 13.47 1.67 9.06 18.00 27.58 36.77 28.33 26.37
Max 6.33 21.10 1.97 103.93 28.93 3.67 15.90 55.50 48.83 49.00 54.63 48.50
Mean 4.95 16.48 1.51 78.06 19.95 2.64 13.46 32.87 37.27 41.94 38.40 38.30
C.D 1.19 1.69 0.36 3.71 1.77 N/A 1.58 13.11 4.57 2.81 4.85 3.16
SE(m) 0.41 0.58 0.12 1.27 0.61 0.39 0.54 4.48 1.56 0.96 1.66 1.08
SE(d) 0.58 0.82 0.18 1.80 0.86 0.55 0.77 6.34 2.21 1.36 2.35 1.53
C.V 14.28 6.08 14.26 2.82 5.25 25.35 6.97 23.63 7.26 3.97 7.48 4.88

Minimum (min), maximum (max), critical difference (cd), standard error (se), coefficient of variance (CV); tiller number/plant (TN/P), flag leaf
length (FLL), flag leaf width (FLW), plant height (PH), peduncle length (PDL), spike number/plant (SN/P), spike length (SL), grain number/
head (GN/H), test weight (TW), days of booting (DOB), days of anthesis (DOA), days of post anthesis (DOPA)

Genetic variability Grain number/head recorded (32.39), (25.95) under stress

High heritability (­ H2%) was recorded in pH (96.81, 96.32);
(98.27, 95.33) and PL (92.64, 90.99); (97.42, 95.06) under
both ambient and heat stress conditions during both the years
2017 and 2018.While the remaining traits showed moderate
to low heritability. The maximum GCV was found in TW
(24.14), PDL (25.82) ambient condition in 2017 and 2018.
conditions in both the years. In 2018, maximum GCV was
observed in peduncle length (25.82), with grain number/
head (25.95) under ambient and stress conditions, respec-
tively. In 2017, (PCV) was higher in spike no./plants (34.21)
and test weight (31.13), while grain number per head and
spike number per plant (39.08, 37.38) under ambient and
heat stress conditions, respectively. During 2018, maximum

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Plant Molecular Biology

Table 4  Selected genotypes Traits Ambient Heat stress Ambient Heat stress
showed minimum and
maximum range of twenty Genotype Min Genotype Min Genotype Max Genotype Max
five (25) and fourteen (14)
genotypes in 2017 and 2018, (a) 2017
respectively years with twelve TN/P HD3059 3 HD3059 3 HD2932 7 PBW722 8
(12) traits under ambient and FLL EC445278 16.17 EC445278 15.23 IC539602 22.97 IC539602 22.97
heat stress condition were
analysed in Rabi season both FLW PBW722 1.63 EC445278 1.67 7HPAN-74 2.73 MP 1 2.73
the years, respectively PH DL788-2 74.37 DL788-2 76.43 IC539602 128.10 IC539602 131.60
PDL PBW722 18.90 HD3059 19.80 IC539221 36.17 MP1 35.53
SN/P IC539221 1.33 HUW468 1.33 DL788-2 3.67 DL788-2 3.33
SL EC576317 7.63 EC576317 8.45 7HPAN-74 15.87 IC539221 15.53
GN/H IC539221 26.67 HD3059 16.89 HUW468 59.33 HUW468 78.67
TW EC445183 20.09 EC445278 23.80 IC539221 69.20 7HPAN-74 73.70
DOB IC539602 38.10 RAJ3765 36.43 IC539531 54.87 IC539531 57.03
DOA RAJ3765 38.93 RAJ3765 37.83 EC445278 58.37 IC539531 56.70
DOPA RAJ3765 36.63 RAJ3765 36.57 IC539531 57.37 IC539531 58.47
(b) 2018
TN/P IC539531 3 IC 539221 3 C-306 6 IC539602 6
FLL HD2932 10.10 C-306 10.60 HD3086 19.17 HD3086 21.10
FLW C-306 1.07 C-306 1.07 HD3086 1.83 HD3086 1.97
PH DL88-2 65.00 DL788-2 64.47 IC145456 104.50 IC539602 103.93
PDL HD2967 13.77 HD2967 13.47 IC539602 30.37 IC145456 28.93
SN/P RAJ3765 2.00 C-306 1.67 HUW206 4.33 HD3086 3.67
SL IC145456 8.94 IC145456 9.06 RAJ3765 14.70 RAJ3765 15.90
GN/H IC539221 18.33 IC539602 18.00 RAJ3765 40.50 RAJ3765 55.50
TW IC539221 21.49 HD2967 27.58 HD3086 48.81 IC539602 48.83
DOB HD2864 35.17 IC539602 36.77 HD2967 47.20 HD2967 49.00
DOA HD3090 25.50 IC145456 28.33 HD3086 54.73 HD3086 54.63
DOPA HD3090 25.10 IC539602 26.37 HD3086 49.50 HD3086 48.50

Minimum (min), maximum (max), critical difference (CD), standard error (SE), coefficient of variance
(CV); tiller number/ plant (TN/P), flag leaf length (FLL), flag leaf width (FLW), plant height (PH), pedun-
cle length (PDL), spike number/plant (SN/P), spike length (SL), grain number/head (GN/H), test weight
(TW), days of booting (DOB), days of anthesis (DOA), days of post anthesis (DOPA)

variation was observed in spike number/plant (27.88), and
grain number per head (35.09) under ambient and heat stress
conditions, respectively. Higher genetic advance (GA) were
found in plant height (23.74, 23.23 and 23.92, 19.99) under
ambient and stress condition in both the years. The genetic
advance value percentage mean (GA% mean) maximum was
observed in test weight and grain no. per head under ambi-
ent and heat stress conditions as observed in 2017. While in
2018 growth season GAVM was higher in peduncle length
as was observed under ambient and heat stress conditions
(Table 5).
The genotypic and phenotypic correlation analyses
In 2017, highly positive and significant correlations were
found between PH (0.613**) to FLL, and DOPA (0.847**)
to DOB (p < 0.01); whereas positive significant correlation
was recorded between PDL to FLL (0.591**) and FLW
(0.575**) (p < 0.01); significant correlations was also deter-
mined between TGW (0.452**) to TN/P, PDL (0.398**)
to PH, TGA under ambient condition. While, in heat stress
condition highly positive significant correlation were found
between DOB (0.941**) to DOA,DOB (0.831**) and DOA
(0.938) to DOPA (p < 0.01), whereas positive significant
correlation showed between FLL (0.591**) to PH. Signifi-
cant correlation revealed between FLL (0.422**) to PDL, SL
(0.440**) to GN/H, and TN/P (0.386**) with (0.410) GN/H
with TW, respectively(p < 0.01) (Table 6a).
In 2018, under ambient conditions, The FLL significantly
correlated with FLW (0.626**) whereas PH was positively
correlated with TN/P (0.433**) (p < 0.01). The TGW was
significantly correlated between TN/P, PL, and GN/H
(0.438**), (0.509**) and (0.558**) (p < 0.01) respectively.
In the case of phenology of chlorophyll at the DOA was pos-
itively correlated with FLW (0.489**) and TGW (0.783**)
while in the DOPA also positively correlated with FLW

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 Plant Molecular Biology

Table 5  Genetic variability of Traits Ambient Heat stress

total twenty five (25) genotypes
2
in 2017 and fourteen (14) H (%) GCV PCV GA GA% mean H2 (%) GCV PCV GA GA% mean
genotypes in 2018, with 12
traits were analysed, in the both 2017
2017 and 2018, respectively TN/P 35.95 16.26 27.12 1.00 20.09 44.51 19.13 28.67 1.28 26.29
FLL 78.08 7.97 9.02 2.88 14.5 87.92 8.53 9.1 3.29 16.48
FLW 85.09 13.84 15.01 0.59 26.31 82.09 12.4 13.69 0.51 23.15
PH 96.81 13.25 13.47 23.74 26.86 96.32 12.98 13.23 23.23 26.24
PDL 92.64 15.14 15.73 9.12 30.01 90.99 13.94 14.61 8.33 27.38
SN/P 47.99 23.71 34.21 0.83 33.82 14.54 14.25 37.38 0.26 11.2
SL 69.63 12.75 15.28 2.86 21.92 72.22 12.18 14.33 2.78 21.32
GN/H 52.72 17.72 24.4 10.41 26.5 68.68 32.39 39.08 21.34 55.29
TW 60.14 24.14 31.13 14.99 38.57 53.13 23.64 32.44 14.69 35.5
DOB 75.12 8.35 9.63 6.84 14.91 75.52 8.45 9.72 6.94 15.12
DOA 72.46 9.24 10.85 7.64 16.19 79.54 8.67 9.72 7.58 15.92
DOPA 85.42 10.65 11.52 9.72 20.27 86.44 9.64 10.36 8.78 18.46
2018
TN/P 46.3 12.46 18.31 0.85 17.47 50.72 14.49 20.34 1.05 21.25
FLL 91.77 18.61 19.42 5.4 36.72 89.52 17.76 18.77 5.7 34.61
FLW 82.59 14.77 16.26 0.37 27.66 48.37 13.81 19.85 0.3 19.78
PH 98.27 14.49 14.61 23.92 29.58 95.33 12.73 13.04 19.99 25.61
PDL 97.42 25.82 26.16 10.44 52.5 95.06 23.06 23.65 9.24 46.31
SN/P 45.53 18.81 27.88 0.76 26.15 22.71 13.74 28.83 0.36 13.49
SL 78.35 12.33 13.93 2.82 22.48 73.12 11.5 13.44 2.73 20.25
GN/H 64.35 22.11 27.56 11.27 36.54 54.67 25.95 35.09 12.99 39.52
TW 70.25 18.02 21.5 11.36 31.12 74.87 12.53 14.48 8.32 22.33
DOB 80.26 8.52 9.51 6.46 15.73 82.41 8.6 9.47 6.75 16.08
DOA 95.97 23.65 24.14 17.33 47.73 88.75 21.02 22.31 15.66 40.79
DOPA 90.25 16.64 17.51 12.07 32.56 92.01 16.56 17.26 12.53 32.71

Analysis of heritability (percent) ­[H2 (%)], genotypic coefficient of variation (GCV), phenotypic coefficient
of variation (PCV), genetic advance (GA), genetic advance value % mean (GA% mean) under ambient and
heat stress condition in Rabi season 2017 and 2018

by (0.400**), GN/H (0.462**), DOB (0.588**), and DOA
(0.708**) (p < 0.01) under ambient conditions. In the case of
heat stress condition the TN/P was positively correlated with
PH, PL, SN/P and GN/H by (0.560**, 0.427**, 0.889**,
and 0.473**) (p < 0.01) respectively. The chlorophyll of the
DOA positively correlate with FLL (0.425**) and booting
stage (0.847**), while in the DOPA correlated with DOB,
and DOA by (0.840**) and (0.895**) (p < 0.01) (Table 6b).
Phenotypic correlation matrix
The TN/P was favorably connected with TGW (0.320**),
and the FLL was highly significant with PH and PDL
(0.540**), (0.501**) respectively, according to the phe-
notypic correlation matrix (Table 7a). The PDL was also
linked to PH (0.394**) and (0.507**) (p < 0.01). NOGPH
and SL had a good relationship (0.315). The DOB had a
strong relationship with DOA and DOPA (0.32** and
0.75**) respectively. Flag leaf length was connected with
plant height and peduncle length (0.489**) (0.352**) and
FLW was correlated with DOA (0.335**) in heat stress con-
ditions. SL was found to have a substantial relationship with
NOGPH (0.415**), DOB (0.724**), DOA (0.668**) and
DOPA (0.802**) (p < 0.01).
In the second year FLL was positively connected with
FLW (0.604**), while NOGPH was positively correlated
with spike length (0.408**). TGW had (0.415**) and
(0.411**) correlations with PDL and NOGPH (Table 7b).
The DOA was shown to be substantially linked with the FLL
(0.344**) FLW (0.425**) and DOB (0.684**) (p < 0.01).
Under ambient environments, the DOPA stage was (0.356**)
(0.448**) (0.561**) and (0.664**) significantly linked with
FLW, NOGPH, DOB, and DOA, respectively. NOGPH was
positively linked with FLW (0.421**) and SL (0.498**)
when subjects were exposed to heat stress. TGW was cor-
related with PH and PDL by (0.599**) and (0.408**) in
heat stress situations, respectively. The DOA was associated
with FLL (0.394**) and DOB (0.677**). All three stages,

13
 Table 6  Genotypic correlation matrix of ambient (lower diagonal) and heat stress (upper diagonal) of twenty four (24) genotypes in 2017, and fourteen (14) genotypes in 2018; with twelve (12)
traits under ambient and heat stress condition were analysed in Rabi season both the years 2017 and 2018, respectively
S. no. TN/P FLL FLW PH PDL SN/P SL GN/H TW DOB DOA DOPA
Plant Molecular Biology

(a) 2017
TN/P 1 0.099 − 0.054 − 0.079 0.059 − 0.026 0.224 0.386** 0.410** 0.233* 0.164 0.264*
FLL 0.096 1 0.236* 0.512** 0.422** − 0.089 − 0.139 − 0.114 0.292* 0.202 0.237* 0.246*
FLW 0.185 0.265* 1 − 0.073 0.332** − 0.233* 0.026 0.044 0.347** 0.230 0.393** 0.315**
PH − 0.017 0.613** − 0.110 1 0.291* 0.018 − 0.176 − 0.191 − 0.103 − 0.256* − 0.233* − 0.221
PDL 0.127 0.591** 0.575** 0.398** 1 − 0.110 0.199 0.091 0.330** − 0.055 − 0.017 − 0.070
SN/P 0.171 − 0.010 − 0.046 − 0.279* 0.014 1 − 0.650** − 0.733** 0.398** 0.223 0.333** 0.211
SL − 0.013 − 0.133 0.088 − 0.322** 0.021 − 0.020 1 0.440** 0.162 − 0.018 − 0.053 − 0.005
GN/H 0.268* − 0.569** 0.103 − 0.486** − 0.397** − 0.087 0.255* 1 − 0.295* 0.112 0.041 0.235*
TW 0.452** 0.306** 0.106 0.243* 0.332** − 0.055 0.183 − 0.627** 1 − 0.105 0.226 0.130
DOB 0.069 0.033 0.240* − 0.541** − 0.215 0.349** − 0.026 0.196 − 0.105 1 0.941** 0.831**
DOA 0.082 − 0.495** − 0.327** − 0.332** − 0.399** 0.118 0.184 0.216 0.122 0.267* 1 0.938**
DOPA 0.032 0.193 0.132 − 0.273* − 0.238* 0.109 0.083 0.022 0.235* 0.847** 0.281* 1
(b) 2018
TN/P 1 − 0.265 − 0.526** 0.560** 0.427** 0.889** − 0.366* − 0.436** 0.473** − 0.033 − 0.112 − 0.263
FLL 0.063 1 0.745** − 0.108 0.352* 0.222 0.144 0.354* − 0.207 0.306* 0.425** 0.386*
FLW − 0.021 0.626** 1 − 0.023 0.541** − 0.139 0.023 0.445** 0.123 0.299 0.306* 0.296
PH 0.433** 0.192 0.034 1 0.732** 0.075 − 0.302 − 0.173 0.722** − 0.416** − 0.485** − 0.687**
PDL 0.311* 0.242 0.395** 0.207 1 0.265 − 0.436** 0.215 0.489** − 0.141 − 0.231 − 0.323*
SN/P − 0.006 0.366* 0.128 − 0.030 0.364* 1 − 0.527** − 0.550** 0.358* 0.070 0.548** 0.212
SL − 0.071 − 0.034 0.202 − 0.588** 0.091 − 0.134 1 0.383* − 0.038 0.187 0.010 0.258
GN/H − 0.063 0.341* 0.101 − 0.323* − 0.032 0.347* 0.351* 1 − 0.193 − 0.120 − 0.177 0.182
TW 0.438** 0.189 0.373* 0.074 0.509** − 0.286 0.362* 0.588** 1 − 0.374* − 0.459** − 0.562**
DOB − 0.059 0.090 0.369* − 0.250 − 0.249 0.299 0.214 − 0.058 − 0.126 1 0.847** 0.840**
DOA 0.317* 0.385* 0.489** − 0.118 − 0.223 0.183 0.346* 0.297 0.158 0.783** 1 0.895**
DOPA 0.300 0.220 0.400** − 0.014 − 0.057 0.268 0.238 0.462** 0.292 0.588** 0.708** 1

Tiller number/plant (TN/P), flag leaf length (FLL), flag leaf width (FLW), plant height (PH), peduncle length (PDL), spike number/plant (SN/P), spike length (SL), grain number/head (GN/H),
test weight (TW), days of booting (DOB), days of anthesis (DOA), days of post anthesis (DOPA)
In bold font, the values equal to 1 because each measure has a perfect linear correlation with itself. The matrix is essentially mirrored from bottom left to top right as the measures are correlated
in both directions

13
 Plant Molecular Biology

Table 7  Phenotypic correlation matrix of ambient (lower diagonal) under ambient and heat stress condition were analysed in Rabi season
and heat stress (upper diagonal) of twenty five (25) genotypes in both the years 2017 and 2018, respectively
2017, and fourteen (14) genotypes in 2018; with twelve (12) traits

S. no. TN/P FLL FLW PH PDL SN/P SL GN/H TW DOB DOA DOPA

(a) 2017
TN/P 1 0.084 − 0.029 − 0.091 0.014 0.092 0.111 0.172 0.210 0.149 0.148 0.198
FLL 0.060 1 0.236* 0.489** 0.352** 0.007 − 0.076 − 0.091 0.192 0.176 0.225 0.227
FLW 0.134 0.245* 1 − 0.045 0.284* − 0.063 0.041 0.033 0.244* 0.145 0.335** 0.276*
PH − 0.036 0.540** − 0.101 1 0.260* − 0.028 − 0.154 − 0.156 − 0.057 − 0.223 − 0.211 − 0.204
PDL 0.065 0.501** 0.507** 0.394** 1 − 0.038 0.134 0.050 0.197 − 0.070 0.017 − 0.050
SN/P 0.079 0.060 − 0.044 − 0.181 0.055 1 − 0.242* − 0.330** 0.041 0.067 0.149 − 0.009
SL − 0.011 − 0.088 0.080 − 0.248* 0.028 0.011 1 0.415** 0.063 0.044 − 0.069 − 0.046
GN/H 0.023 − 0.393** 0.043 − 0.369** − 0.276* 0.010 0.315** 1 − 0.281* 0.171 − 0.001 0.148
TW 0.320** 0.269* 0.133 0.186 0.219 − 0.072 0.095 − 0.374** 1 − 0.064 0.213 0.104
DOB 0.133 − 0.001 0.163 − 0.476** − 0.193 0.211 0.019 0.173 − 0.075 1 0.724** 0.668**
DOA 0.170 − 0.339** − 0.261* − 0.293* − 0.321** 0.161 0.109 0.145 0.061 0.320** 1 0.802**
DOPA 0.033 0.145 0.102 − 0.262* − 0.207 0.060 0.100 0.049 0.087 0.750** 0.299* 1
(b) 2018
TN/P 1 − 0.104 − 0.145 0.382* 0.314* 0.394** − 0.175 − 0.068 0.392* 0.024 − 0.139 − 0.211
FLL 0.054 1 0.459** − 0.120 0.294 0.055 0.115 0.227 − 0.157 0.256 0.394** 0.350*
FLW 0.009 0.604** 1 0.006 0.350* − 0.017 0.135 0.421** 0.024 0.237 0.159 0.170
PH 0.292 0.184 0.029 1 0.701** 0.071 − 0.226 − 0.074 0.599** − 0.348* − 0.471** − 0.638**
PDL 0.241 0.221 0.335* 0.200 1 0.141 − 0.348* 0.219 0.408** − 0.145 − 0.226 − 0.309*
SN/P − 0.127 0.268 0.077 − 0.052 0.207 1 − 0.253 − 0.219 0.060 0.075 0.261 − 0.023
SL 0.034 0.029 0.197 − 0.510** 0.105 − 0.074 1 0.498** 0.057 0.135 0.010 0.204
GN/H 0.011 0.318* 0.139 − 0.250 − 0.017 0.106 0.408** 1 − 0.067 − 0.085 − 0.157 0.131
TW 0.144 0.151 0.267 0.050 0.415** − 0.026 0.279 0.411** 1 − 0.324* − 0.330* − 0.439**
DOB 0.008 0.106 0.354* − 0.204 − 0.224 0.218 0.284 0.073 − 0.100 1 0.677** 0.716**
DOA 0.234 0.344* 0.425** − 0.122 − 0.223 0.145 0.282 0.214 0.128 0.684** 1 0.835**
DOPA 0.270 0.225 0.356* − 0.004 − 0.041 0.155 0.253 0.448** 0.235 0.561** 0.664** 1

Tiller number/plant (TN/P), Flag leaf length (FLL), Flag leaf width (FLW), Plant height (PH), Peduncle length (PDL), Spike number /plant
(SN/P), Spike length (SL), Grain number/head (GN/H), Test weight (TW), Days of booting (DOB), Days of anthesis (DOA), Days of post anthe-
sis (DOPA)
**Significant at 0.01 level of probability, *significant at 0.05 level of probability
In bold font, the values equal to 1 because each measure has a perfect linear correlation with itself. The matrix is essentially mirrored from bot-
tom left to top right as the measures are correlated in both directions

DOB, DOA, and DOPA, were linked by chlorophyll content.

Under heat stress, the DOPA stage was favorably linked with Table 8  Distribution of TaSKP1 like genes across the wheat chromo-
DOPA (0.716**) and DOA (0.835**) (p < 0.01). somes
Chromosome no. Sub-genome A Sub-genome B Sub-
RNA isolation, cDNA synthesis and TaSKP1 genome
amplification from different wheat genotypes D

1 1 1 2
Total RNA was isolated from flag leaf and ear head tissues
2 4 2 6
of the genotypes at the anthesis stage both from the ambient
3 3 8 8
and HS exposed group of plants and cDNA was synthesized.
4 3 2 1
Ten different SKP1 like full coding sequence variants of
5 8 9 10
520 bp and 350 bp were amplified, ligated into the pGEM-T
6 3 4 5
Easy vector, sequenced, structurally analyzed and deposited
7 5 5 5
in the NCBI nucleotide database with the following acces-
Total 27 31 37
sion numbers (Table 8).All the TaSKP1sequences carried

13
Plant Molecular Biology
two conserved domains from the BTB (98–172 amino acids)
and SKP1 (10 to 124 amino acids) super families. A Clustal
omega multiple sequence alignment was performed with
the ten isolated sequences along with a range of additional
available SKP1 sequences (Fig. 3). These result confirmed
isolated sequence as SKP1.
Expression pattern of TaSKP1‑6B‑4 genes
in different genotypes under ambient and heat
stress exposure conditions
Quantitative RT-PCR analysis was done on RNA iso-
lated from flag leaves and spikes at the inflorescence or
anthesis stage under ambient and high temperature expo-
sure to assess the expression of TaSKP1-6B-4 in wheat
(Fig. 2). As a reference gene, the internal housekeep-
ing gene 18SrRNA was employed. For the relevant heat
stress expression analysis, the ambient assessment was
considered as 1, and compared with stress values of each
cultivar to examine the relative expression levels in flag
leaf and ear head under ambient and high temperature
conditions. The results revealed a differential expres-
sion pattern that was substantially elevated, moderately
Fig. 2  Expression patterns of SKP1 transcripts in wheat genotypes
in response to ambient and high temperature condition from flag
leaf and ear head. In relative transcript expression profiling, ambient
temperature value was taken as 1 and relative fold changes in expres-
sion were calculated for the respective heat stress value. Fold change
is shown on a log2 scale from high to low expression of each SKP1
up-regulated, and down-regulated in different tissues
and temperature regimes. The examination of the qRT-
PCR findings revealed that the each chosen genotype had
significantly altered expression in heat stress treatments
compared to ambient conditions. HD2967 and IC145456
had the highest expression (9.9-fold) and (6.18-fold)
compared to the ambient plants in the flag leaf heat stress
conditions (Fig. 2a). C-306 (2.15), RAJ3765 (2.36), and
HD3090 (3.0) fold expression was found to be moderate.
In contrast, several cultivars, such as HD 2864, DL-788-
2, WR544, HD2932, and HD3086, were shown to down-
regulate expression under stress conditions by 0.81, 1.22,
1.26, 1.57, and 0.16 fold, respectively. On the other hand
in ear head expression did not exhibit the same pattern as
flag leaf expression, showing high up regulation, in the
cv. C-306 (15.88-fold), RAJ3765 (8.37-fold), and HD2932
(8.25-fold) cultivars with the greatest levels of expression.
DL-788-2, WR544, HD3086, and HD2967 were all found
to be down regulated by 1.11, 0.19, 0.29, and 2.28 fold,
respectively (Fig. 2b). HD3090 (4.40-fold), IC145456
(4.38-fold), and HD2864 (3.60-fold) expression patterns,
on the other hand, were modestly expressed.
transcripts. a Relative transcript expression profiling in flag leaf.
b Relative transcript expression profiling in ear head c. Heat map
The color scale represents increased (red) and decreased (blue) fold
change in the levels of TaSKP1-6B-4 transcripts in different tissues
(flag leaves and ear head) when exposed to heat stress as compared to
the respective levels in the controls for cultivar
13

Fig. 3  Phylogenetic tree of SKP1-like genes isolated from wheat gen-
otypes. Multiple sequence alignment of the 10 TaSKP1-6B-4 amino
acid sequences with other monocot species SKP1s using the Neigh-
bor-Joining method with 1000 bootstrap replications
Genomic distribution of the TaSKP1‑like gene family
A total of 95 TaSKP1 like genes were identified in wheat
genome with HMMER search using HMM profiles of
SKP1 like proteins obtained from Pfam database as a
query sequences. The presence of a well-conserved SKP1
like domain is confirmed by NCBI-Conserved Domain
Database searches (Fig. S1). The TaSKP1 proteins with
no conserved SKP1 domain were excluded from our
analysis. The predicted TaSKP1 proteins were named as
TaSKP1-1 to TaSKP1-95, as per their positions on wheat
chromosome numbers 1–7 of genomes A, B, and D respec-
tively (Table S2). Accordingly the SKP1 member isolated
and analyzed in this study was named as TaSKP1-6B-4,
(TraesCS6B02G004300) located on chromosome 6B and
fourth in series in this chromosome. The closest homeo-
logues to this SKP1 gene member were determined as
TraesCS6A02G000300 and TraesCS6D02G001600. The
length of TaSKP1 like proteins ranged between 134 to
558 amino acids with an average of 221amino acids. Out
of the 95 predicted TaSKP1 like proteins, only one protein
sequence (TaSKP1-17) contained two SKP1 like domains,
rest of the protein sequences contained only one SKP1
like protein domain. The molecular mass of the predicted
13
Plant Molecular Biology
TaSKP1 like proteins ranged from 14,620.48 to 62, 272.41
kDa and also showed a broad range of isoelectric points
ranging from basic (4.18 pKa) to acidic (9.33 pKa). The ali-
phatic index indicates the thermostability of proteins, which
ranged from 65.13 to100. The TaSKP1-55 was found to be
the most thermostable among the predicted TaSKP1 like
proteins (Table S2).
Phylogenetic distribution of wheatTaSKP1
like proteins
The evolutionary analysis was carried out based on the
amino acid sequences of the cloned TaSKP1 like proteins
and between other known SKP1 sequences. The evolu-
tionary relationship with SKP1s identified from different
gramene family species was carried out using the Neigh-
bor-Joining method with 1000 bootstrap replications. The
results showed that Triticum aestivum (AY316293) as very
closely related with Aegilops tauschii (XM020326669) and
TaHD3086-SKP1-6B-4 amongst all the wheat genotypes
was distantly related with Phyllostachys edulis (FP100547);
whereas Phyllostachys edulis also showed a distant relation-
ship to HD2932 as shown in Fig. 3. TaSKP1-4B-4 protein of
cvHD2932 was found to be distantly related with HD3086.A
phylogenetic analysis was also performed based on the
amino acid sequences of TaSKP1 like genes using SMART
genomics web tools, after the domain sequences of total 95
TaSKP1 were identified. The distribution of TaSKP1 was
divided into 11 subfamilies, and a phylogenetic tree was con-
structed with the amino acid sequences of other monocot
gramene family species. Based on domain phylogeny tree,
the Triticum aestivum was very closely related with Triticum
uratu and Aegilops tauschii. The phylogenetic distribution of
conserved motifs had been detected across the TaSKP1 like
gene family sequences. The phylogenetic analysis revealed
that SKP1 gene members of the same family made a cluster
with their respective orthologs. The distribution of SKP1
gene amongst other monocots analysed maximum were
Aegilops tauschi 31 and Panicum virgatum 27 whereas mini-
mum observed in Hordeum vulgare 5 and Brachypodium
distachyon 9, SKP1 genes. In the Leersia perrieri 14 SKP1
genes were present and one smart ring box domain which
was not seen in other monocots (Fig. 4).
Chromosomal organization of TaSKP1 like gene
Using sequence homology and an HMM-based approach,
we identified 95TaSKP1 encoding genes in three wheat
sub genomes A, B and D. The chromosomal distribution
of TaSKP1likegenes in the wheat A, B, and D genome are
depicted in Fig. S2. Each chromosome of every sub genomes
contained at least one TaSKP1like gene. However, the distri-
bution of TaSKP1 like genes between the chromosomes was
Plant Molecular Biology
Fig. 4  Phylogenetic analysis of wheat SKP1 like genes with other
monocot family members using maximum likelihood method show-
ing a syntenic relationship between Triricumaestivum and its progeni-
tors. Zea mays (Zm), Sorghum bicolour (Sb), Panicum virgatum (Pv),
not equal. The maximum numbers of TaSKP1 genes were
present in the chromosomes 5D (10) followed by 5B (9), 5A
(8), 3B (8), and chromosome 3D (8) while chromosome 1A,
1B, and 4D contained only singleTaSKP1 gene. Maximum
number of TaSKP1 like genes were present in sub-genome
D followed by sub-genome B and A (Table 8). In addition,
a gene cluster of TaSKP1-13, TaSKP1-14, TaSKP1-15, and-
TaSKP1-16 like genes was found at the chromosome number
2D, and a similar gene cluster of TaSKP1-87, TaSKP1-88,
TaSKP1-89, andTaSKP1-90 like genes was also present at
the chromosome 7B. As a whole, we found that chromosome
number 5 had the highest number of TaSKP1 like genes at
27, followed by chromosome number3 (19), chromosome
number. 7 (15), chromosome number 2 (12) and chromo-
some number 6 (12) (Table 8).
Exon–Intron organization and motif composition
of wheat SKP1 like proteins
The distribution of exon–intron was analyzed to gain more
insight into the structural features of TaSKP1 like genes. The
number of exon and intron varies from one to seven among
Setaria italica (Si), Oryza sativa (Os), Leersia perrieri (Lp), Brachy-
podium distachyon (Bd), Hordeum vulgare (Hv), Aegilops tauschii
(Aet), and Triticum urartu (Tu)
TaSKP1 like genes (Fig. S3). Among the 95TaSKP1 like
genes, 40 genes were intron less while 41 genes had only
one intron, 4 TaSKP1 like genes contained three introns, 4
TaSKP1 like genes harbored three introns, 2 TaSKP1 like
genes contained seven introns, and only 1 TaSKP1 like
gene contained five and six introns respectively (Fig. S3).
For better understanding, the diversity of TaSKP1 like pro-
tein motifs, the conserved motif analysis of TaSKP1 like
protein sequences were investigated by using the online
server MEME (http://​meme.​nbcr.​net/​meme/​cgi-​bin/​meme.​
cgi) (Fig. 5). From the total 10 predicted motifs, motif 1
containedTaSKP1 like protein domain which is the unique
feature of all TaSKP1 like proteins.
Cis‑regulatory element analysis in the upstream
promoter region of wheat SKP1 like genes
Cis-regulatory elements are the binding sites of transcription
factors responsible for transcriptional regulation of the gene.
Usually, the regulatory region is restricted to the 5′ upstream
(promoter) sequence of the gene (Singh et al. 2019). Thus,
2 K upstream regulatory (proximal promoter) region from all
13
 Plant Molecular Biology

Fig. 5  Schematic distribution of 10 conserved motifs predicted in 95 SKP1 like proteins from wheat using the MEME software tool (http://​
meme-​suite.​org)

13
Plant Molecular Biology
the 95 TaSKP1 like genes was extracted and used to survey
stress-responsive regulatory elements by using the online
database PlantCARE. The 2000 bp upstream promoter
region of 95 TaSKP1 family members in wheat contains
a large number of core elements (TATA-boxes and CAAT-
boxes) and light-responsive elements. In addition, there were
various components associated with growth and develop-
ments, hormone responses, vis. TGACG motifs, CGTCA
motifs, TATC-boxes, ABREs, TGA elements, and TCA ele-
ments, and stress-related components MBSs, AREs, MRE,
LTRs, TCT-motif, G-box, Sp1, GT1-motif and TC-rich
repeats (Fig. S4).
Three dimensional structure prediction of wheat
SKP1 like proteins and Ramachandran plot
analysis the interactions of the glycine, pre‑proline
and proline in TaSKP1‑6B‑4 protein structure
The 3D protein structure of TaSKP1 like proteins was
predicted using the SWISS-MODEL online server. Selec-
tion of the perfect 3D model was based on their respec-
tive C-score value. Out of 525 amino acid residues, 175
residues occurred in the most favored region in the refer-
ence TaSKP1 protein. To build the 3D model, the crystal
structure of TaSKP1 sequence identity with AY316293.1
Triticum aestivum was used as a template and QMEAN
score was − 2.09 (Table S3). Also, the modeled structure
was validated by predicting the Ramachandran plot which
indicated 88.44% in the favored region. The structure
showed high similarity with 6B chain of Oryza sativa
SKP1 protein. Ramachandran plot was used for the vali-
dation of modeled protein structure (Fig. 6). MolProbity
score is a combined protein quality score that gives the
idea of crystallographic resolution at which such quality
would be expected. In an ideal case, it should be as low as
Fig. 6  Ramachandran plot for the TaSKP1(cvHD2967) gene obtained
from Swiss Model. a Schematic representation of TaSKP1-HD2967
gene. b 3D structure model: red, blue and orange color represents
possible. MolProbity score was 2.19 and clash score was
identified as5.70.
The Ramachandran plots visualize the dihedral angles
of the polypeptide backbone (ψ and ϕ) in proteins. Each
point on the Ramachandran plot represents one amino
acid. In a polypeptide, the backbone bonds rotate relatively
freely. These rotations are represented by torsion angles
Phi (ϕ) and Psi (ψ), respectively. White areas indicate con-
formations where polypeptide atoms are closer than the
sum of their Van Der Waal’s radiuses. Except for glycine,
which does not have a side chain, these regions are satiri-
cally forbidden. In the dark green areas, the amino acids
can arrange themselves in specified ways without steric
collisions, referring to the α-helix and β-helix conforma-
tions (Fig. 7). If Van Der Waals radii are slightly shorter,
light green areas indicate that atoms can be moved closer
together when their Vander Waals radii are slightly shorter.
There is an additional region that corresponds to the left
α-helix. A more detailed analysis was performed by mod-
eling Ramachandran plots for glycine, preproline, and pro-
line. Here, we analyzed the most distinctive feature of the
glycine Ramachandran plot—the tendency for ψ to cluster
near 180° and 0°. We focused on the ψ dependent interac-
tions (Fig. 7). For each interaction, we first calculated the
model curve of the corresponding inter-atomic distance as
a function of ψ. In glycine, the ψ angle is typically clus-
tered at ψ = 180° and ψ = 0°. We showed that these clus-
ters correspond to conformations by β sheet interactions by
126, 77, 83, 141 and 48 (Table S4) whereas proline were
93, 144, 159, 52, 90, 74, and 54. The results for glycine
were identical to that of the generic Ramachandran plot.
helix, beta strand and coil. c Alignment of amino acid sequences. d
Model building result using QMEAN as a quality measure to guide
the modeling processes
13

Fig. 7  Schematic representation and comprehensive overview of TaSKP1-HD2967 amino acid interactions. a Glycine, b proline, c pre-proline
Discussion
With the adverse attributes of climate change a reality, wheat
genotypes able to resist terminal heat stress is the need of the
hour to transition into sustainable agriculture and land use.
We need plants able to tolerate extreme climate changes to
meet the growing rise in population and food and nutritional
demand as well as with high productivity. A diverse con-
trasting panel of wheat germplasm was selected to analyze
genetic variability and involvement of UPS/SKP1 during
anthesis stage heat stress exposure in field based in natura
environmental conditions. Evolutionary biology shows that
genetic and gene expression diversity shapes adaptation to
extreme environment (Yu et al. 2022). Mechanistic under-
standing of terminal heat stress tolerance in a tolerant cul-
tivar will aid in rational designing of tolerant and climate
smart wheat for future.
Senescence and agronomic traits as indicator
of anthesis stage heat stress tolerance
Contrasting phenology and growth habit of wheat culti-
vars showed remarkable positive and negative variations
in the anthesis stage affecting yield and plant health. The
combined analysis of variance indicated significant differ-
ences amongst the genotypes for all the characters studied
in both the environmental conditions. In the first year study,
the ambient raised plants showed normal yield but some
traits showed increased expression after exposure to heat
stress. The loss of chlorophyll is associated with senescence
and stay-green genotypes are better able to maintain photo-
synthesis, resulting in a high percentage of tolerability and
superior grain quality compared to the regular plants. Our
results showed that cv. RAJ3765, HD2967 and HD3086
maintained chlorophyll content at booting, anthesis and post
anthesis stages prolonging the stay-green time in both the
13
Plant Molecular Biology
years under heat stress. The stay-green trait is antagonistic
to senescence with chlorophyll and photosynthetic capacity
of leaves maintained or prolonged, which is considered an
indicator of heat tolerance (Fokar et al. 1998; Yang et al.
2014; Jing et al. 2020). In the present scenario, maintaining
grain yield is a fundamental concern in breeding, which can
be achieved by prolonged stay green capacity of the plants
and thereby resist heat stress injury under heat stress (Liu
et al. 2007; Jing et al. 2020).
Our study dealt with finding terminal heat stress tolerant
genotype which can overcome adverse impact of flowering
and grain-filling stage heat wave. Various manifestations of
physiological traits contribute towards this response diver-
gence. We observed highest contribution in manifestation of
genetic divergence exhibited by grain yield and chlorophyll
contents in both the years of investigations. Grain number
per plant was highest in case of HUW 468 in the first year
post heat stress, whereas in the second year this genotype
was replaced by RAJ3765 (Table 4). HUW468 is a reported
heat susceptible genotype which interestingly showed high
grain number per head in the first year, however in the sec-
ond year it could not show the same high yield. This could
be attributed to a boost in metabolism by a short stint of
high temperature. The results revealed that plant height and
grain yield have contributed maximum to divergence and
should form basis of selection for genotypes. The associa-
tion studies of maximum and minimum indicated that the
grain yield of wheat can be improved by selecting germ-
plasm having higher performances for these traits. Many
of the traits exhibited analysis of ­[H2 (%)], (GCV), (PCV),
(GA) and (GA %mean) under ambient and heat stress con-
dition in both the years consistently. Thus, these traits are
amenable to the phenotypic selection through coefficient of
phenotypic and genotypic variation, for superior wheat culti-
vars with improved wheat productivity (Table 5). High herit-
ability was found in PH, followed by PDL, and Chlorophyll
Plant Molecular Biology
(DOB, DOA, and DOPA). In a study carried out by Sahu
et al. (2005), high heritability for total number of effective
tillers per plant, spike length, number of grain per spike and
TGW were reported. Moderate to low heritability for grain
yield were also reported by other workers (Pathak and Nema
1985; Belay et al. 1993).
Thousand grain weights determine grain number and
grain yield in wheat. Genotypic and phenotypic correlation
interaction analysis results (Tables 6, 7) showed a significant
variation among the genotypes in the number of tillers ­hill−1,
along with thousand grain weight. Total number of effective
tillers, spike length and grain yield per plant, number of
spikelets per ear head, number of grains per spike, TGW and
chlorophyll contents showed significant value of phenotypic
and genotypic co-efficient of variation under ambient and
heat stress in both the seasons. It also exhibited considerable
diversity in the chlorophyll content at booting anthesis and
post anthesis stages under ambient and high temperature
conditions amongst all the traits.
Transcript profiling of TaSKP1‑6B‑4 and genome
wide analysis of wheat SKP1 like gene family
Differential relative expression of TaSKP1-6B-4 gene was
observed in two different tissues of flag leaf and ear head
under heat stress conditions over ambient temperature. Ear-
lier we had reported that, TaSKP1-6B-4 gene is induced by
terminal heat stress in flag leaf and ear head of cv. HD2967
(Jaiswal et al. 2022). Here, we observed that the TaSKP1-
6B-4 transcript expression level of ear head tissues was
higher than that of flag leaf tissues under heat stress over
controlled plant. However, large variations in transcript
level changes were detected for TaSKP1-6B-4 genes in dif-
ferent cv. viz. C-306, HD2932 and RAJ3765 in ear head and
HD2967 and IC145456 in flag leaf tissues respectively. A
heat map was constructed based on the qRT-PCR expression
analysis in the various plant tissues. Although the TaSKP1-
6B-4 gene shares high level of similarities, each TaSKP1-
6B-4 from different genotypes showed differential expres-
sion in the heat map. As key proteins in the UPS signalling
pathway, SKP1-like genes play a major role in the recruit-
ment of the target proteins to be guided to proteasome 26S
in plant development processes and in response to stressors
(Vierstra 2003, 2009; Stone and Callis 2007; Hua and Vier-
stra 2011; Stone 2014; Melo et al. 2021; Hua 2023).
In the ubiquitin proteasome system, SKP1 is a core
subunit of E3 ubiquitin ligases. An earlier study reported
six SKP1 like genes from wheat (Hong et al. 2013); sub-
sequently a whole genome analysis by El Beji et al. (2019)
documented presence of 92 SKP1 like genes from wheat. In
other plant species A. thaliana 21 SKP1, rice 31 SKP1-like
genes had been reported (Kong et al. 2004; Kahloul et al.
2012). The chromosomal organization of the 95 TaSKP1 like
encoding genes is distributed unevenly in the three wheat
sub genomes A, B and D. The aliphatic index indicates the
thermo stability of proteins and the TaSKP1-55 was found
to be the most thermostable among the predicted TaSKP1
like proteins. The SKP genes were distributed among 21
chromosomes and most showed high sequence homology
with SKP genes located on the homeologous chromosomes
which could be attributed to the allohexaploidy nature of the
wheat genome. The phylogenetic tree showed the clustering
of Triticum aestivum TaSKP1-6B-4 with other monocot spe-
cies orthologs based on a domain phylogeny. As expected
a very close relationship with Triticum uratu and Aegilops
tauschi, the progenitors of bread wheat and contributor of
A and D genome was observed. Wheat cvHD3086 TaSKP1-
6B-4 showed very close identity with earlier reported wheat
SKP1 (AY316293), Aegilops tauschii (XM020326669) and
was found to be distantly related to Phyllostachys edulis.
Cis-regulatory elements are the binding sites of tran-
scription factors responsible for transcriptional regulation
of the gene. Usually, the regulatory region is restricted to
the 5′ upstream (promoter) sequence of the gene (Singh
et al. 2019). Besides the fundamental common cis regula-
tory elements (CRE), the upstream sequence of SKP1 genes
harbored diverse CREs. Several A-box, MSA-like element,
WUN-motif, MBSI and MBS element were found to be
related to growth and development. Additionally, differ-
ent elements related to the hormone response viz. TGACG
motifs, CGTCA motifs, TATC-boxes, ABREs, TGA ele-
ments, and TCA elements, and stress-related components
viz. MBSs, AREs, MRE, LTRs, TCT-motif, G-box, Sp1,
GT1-motif and TC-rich repeats. The 3D protein structure
of TaSKP1 like proteins was predicted using the SWISS-
MODEL online server and selection of the perfect 3D
model was based on their respective C-score value. Out of
525 amino acid residues, 175 residues occurred in the most
favoured region in the reference TaSKP1 protein. The crystal
structure of TaSKP1-6B-4 showed 58.97% sequence iden-
tity with the wheat reference SKP1 sequence AY316293.1,
available at NCBI nucleotide database. Analysis of cis-reg-
ulome in plants will provide information on how cis regu-
lating DNA sequences function to coordinate responses to
environmental and developmental stimuli (Schmitz et al.
2022). Complex pattern of gene expression regulation can
be answered by identification and characterization of cis ele-
ments. Genome editing of promoter elements will comple-
ment other efforts to improve plant trait improvement.
Under abiotic stress, SKP1-6B-4 modulations appear
to play an important role in plant physiological functions
and defence responses. Adequate genetic variability was
also found to be present in the genotypes studied here.
The separation and selection of varieties based on high
heritability of intended traits make it easy for breeders to
utilize this knowledge and implement this information in
13

transgressive segregation breeding programme. Although
high heritability is not alone responsible in selecting
superior varieties, coupling it with high genetic advance
as well as genotypic and phenotypic variation will make it
more effective. Amongst the genotypes analysed HD2967,
HD3086 and WR544 showed maximum potential for
incorporation in pre breeding programs for developing
late stage or grain filling stage high temperature stress
tolerance. RAJ3765, a well-known heat check performed
as expected showing positive response in the first year.
The result of field based experiments is close to the real
world setting versus controlled laboratory environment
and offers contextual data on different interactions hap-
pening in the natural environment. Thus our results also
throw a more realistic picture of climate change and rise
in ambient temperature in a field setting and possible
mechanisms we can pursue to combat this unavoidable
adversity.
A schematic diagram outlining the total sequence of
experiments carried out on genetic variability, TaSKP1-
6B-4 RNA expression under terminal heat stress in field
Fig.8  A schematic diagram showing the sequence of experiments and outcomes of the 2 year field based characterization of genetic variability
and SKP1transcript profiling under terminal heat stress in wheat
13
Plant Molecular Biology
environment and whole genome analysis of SKP1 gene
family from wheat is illustrated in Fig. 8.
Conclusions
Terminal heat stress tolerance in wheat was analysed and
positively responsive genotypes were identified from a panel
of diverse germplasm through characterization over a period
of two consecutive years under field conditions by expos-
ing the plants to high ambient temperature. The modulation
of heat stress response by UPS was also analysed through
whole genome analysis and transcript heterogeneity profiling
of TaSKp1-6B-4, an integral component of ubiquitination
mediated protein turnover. The potential functional involve-
ment of these post translational modifications was evident
and can be further dissected mechanistically in the best
genotype identified from this study, cvHD2967, in terminal
heat stress tolerance. Selected genotypes can be utilized in
crop improvement and breeding programs for development
of wheat varieties exhibiting heat tolerance at the termi-
nal growth stage through conventional as well as new age
technologies.
Plant Molecular Biology
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 11103-0​ 23-0​ 1389-7.
Acknowledgements The work described here was carried out under the
aegis of ICAR-NIPB in- house project, NRCPB-RPP-2017-2021/02 of
SB. PJ acknowledges Indian Council of Agricultural Research, National
Project on Functional Genomics and Genetic Modification (ICAR-
NPFGGM) for a research fellowship.
Author contributions SB conceived, conceptualized and designed the
experiments. PJ carried out all the field and laboratory experiments
including phenotyping, SKP1 sequence isolation and cloning. KT car-
ried out phylogenetic and protein structure analysis and AS performed
all the other bioinformatic analyses. KB drafted the bioinformatics part
of the manuscript. ANS provided valuable inputs and contributed to
data analysis. PJ and SB drafted, edited and finalized the manuscript.
All authors have read and approved the manuscript.
Funding No funding was received for this study.
Data availability The sequence data and accession numbers mentioned
here can be found on the NCBI website at (https://​www.​ncbi.​nlm.​nih.​
gov).
Declarations
Conflict of interest On behalf of all authors the corresponding author
states that there is no conflict of interest.
References
Adams EHG, Spoel SH (2018) The ubiquitin–proteasome system
as a transcriptional regulator of plant immunity. J Exp Bot
69(19):4529–4537. https://​doi.​org/​10.​1093/​jxb/​ery216
Asseng S, Ewert F, Martre P, Rötter RP, Lobell DB, Cammarano D et al
(2015) Rising temperatures reduce global wheat production. Nat
Clim Chang 5(2):143–147. https://​doi.​org/​10.​1038/​nclim​ate24​70
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren
J, Li WW, Noble WS (2009) MEME SUITE: tools for motif dis-
covery and searching. Nucleic Acids Res 37(Web Server):W202–
W208. https://​doi.​org/​10.​1093/​nar/​gkp335
Belay G, Tesemma T, Becker HC, Merker A (1993) Variation and inter-
relationships of agronomic traits in Ethiopian tetraploid wheat
landraces. Euphytica 71:181–188
Bennett D, Reynolds M, Mullan D, Izanloo A, Kuchel H, Langridge P,
Schnurbusch T (2012) Detection of two major grain yield QTL in
bread wheat (Triticum aestivum L.) under heat, drought and high
yield potential environments. Theor Appl Genet 125(7):1473–
1485. https://​doi.​org/​10.​1007/​s00122-​012-​1927-2
Bokshi AI, Tan DKY, Thistlethwaite RJ, Trethowan R, Kunz K (2021)
Impact of elevated ­CO2 and heat stress on wheat pollen viability
and grain production. Funct Plant Biol 48(5):503. https://​doi.​org/​
10.​1071/​fp201​87
Castelán-Muñoz N, Herrera J, Cajero-Sánchez W, Arrizubieta M, Trejo
C, García-Ponce B, Garay-Arroyo A (2019) MADS-Box genes are
key components of genetic regulatory networks involved in abiotic
stress and plastic developmental responses in plants. Front Plant
Sci. https://​doi.​org/​10.​3389/​fpls.​2019.​00853
Chatzopoulos T, Domínguez IP, Toreti A, Adenäuer M, Zampieri M
(2021) Potential impacts of concurrent and recurrent climate
extremes on the global food system by 2030. Environ Res Lett
16(12):124021. https://​doi.​org/​10.​1088/​1748-​9326/​ac343b
Dutta M, Phogat BS, Kumar S, Kumar N, Kumari J, Pandey AC et al
(2015) Development of core set of wheat (Triticum spp.) germ-
plasm conserved in the national Genebank in India. In: Advances
in wheat genetics: from genome to field: proceedings of the 12th
international wheat genetics symposium. Springer, Japan, pp
33–45. https://​doi.​org/​10.​1007/​978-4-​431-​55675-6_4
Djanaguiraman M, Boyle DL, Welti R, Jagadish SVK, Prasad PVV
(2018) Decreased photosynthetic rate under high temperature
in wheat is due to lipid desaturation, oxidation, acylation, and
damage of organelles. BMC Plant Biol. https://​doi.​org/​10.​1186/​
s12870-​018-​1263-z
El Beji IH, Mouzeyar S, Bouzidi MF, Roche J (2019) Expansion and
functional diversification of SKP1-like genes in wheat (Triticum
aestivum L.). MDPI. https://​doi.​org/​10.​3390/​ijms2​01332​95
Fokar M, Nguyen HT, Blum A (1998) Heat tolerance in spring wheat
I. Estimating cellular thermo tolerance and its heritability.
Euphytica. https://​doi.​org/​10.​1023/A:​10183​46901​363
Frieler K, Lange S, Piontek F, Reyer CP, Schewe J, Warszawski L,
Zhao F, Chini L, Denvil S, Emanuel K, Geiger T (2017) Assess-
ing the impacts of 1.5 C global warming–simulation protocol
of the Inter-Sectoral Impact Model Inter comparison Project
(ISIMIP2b). Geo Sci Model Dev 10:4321–4345
Hanson WD (1956) Heritability, statistical genetics and plant breed-
ing. NASNRC 981. The National Academies Press, Washington,
pp 115–140. https://​doi.​org/​10.​17226/​20264
Hong MJ, Kim DY, Seo YW (2013) SKP1-like-related genes interact
with various F-box proteins and may form SCF complexes with
Cullin–F-box proteins in wheat. Mol Biol Rep 10:1007
Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G (2014) GSDS 2.0:
an upgraded gene feature visualization server. Bioinformat-
ics 31(8):1296–1297. https://​doi.​org/​10.​1093/​bioin​forma​tics/​
btu817
Hua Z (2023) Deciphering the protein ubiquitylation system in plants.
J Exp Bot. https://​doi.​org/​10.​1093/​jxb/​erad3​54
Hua Z, Vierstra RD (2011) The Cullin-RING ubiquitin-protein ligases.
Annu Rev Plant Biol 62(1):299–334. https://​doi.​org/​10.​1146/​
annur​ev-​arpla​nt-​042809-​112256
Jackson PK, Eldridge AG, Freed E, Furstenthal L, Hsu JY, Kaiser BK,
Reimann JD (2000) The lore of the RINGs: substrate recognition
and catalysis by ubiquitin ligases. Trends Cell Biol 10(10):429–
439. https://​doi.​org/​10.​1016/​s0962-​8924(00)​01834-1
Jaiswal P, Sahi AN, Barthakur S (2022) Cytokinin seed priming medi-
ated induction of terminal heat stress tolerance and expression
profiling of SKP1 transcripts, a component of ubiquitin protea-
some system in bread wheat (Triticum aestivum L.): a transgen-
erational analysis. Plant Growth Regul 98(2):259–280. https://d​ oi.​
org/​10.​1007/​s10725-​022-​00861-6
Jing J, Guo S, Li Y, Li W (2020) The alleviating effect of exogenous
polyamines on heat stress susceptibility of different heat resistant
wheat (Triticum aestivum L.) varieties. Sci Rep. https://​doi.​org/​
10.​1038/​s41598-​020-​64468-5
Kahloul S, HajSalah El Beji I, Boulaflous A, Ferchichi A, Kong H,
Mouzeyar S, Bouzidi MF (2012) Structural, expression and inter-
action analysis of rice SKP1-like genes. DNA Res 20(1):67–78.
https://​doi.​org/​10.​1093/​dnares/​dss034
Khomdram S, Barthakur S (2015) In situ evaluation of high tempera-
ture stress in wheat (Triticum aestivum L.)—validation of a port-
able heat trap chamber. Ann Agric Res 36(2):123–131
Kong H, Leebens-Mack J, Ni W, dePamphilis CW, Ma H (2004) Highly
heterogeneous rates of evolution in the SKP1 gene family in plants
and animals: functional and evolutionary implications. Mol Biol
Evol 21(1):117–128. https://​doi.​org/​10.​1093/​molbev/​msh001
Kong H, Landherr LL, Frohlich MW, Leebens-Mack J, Ma H,
DePamphilis CW (2007) Patterns of gene duplication in the
plant SKP1 gene family in angiosperms: evidence for multiple
13

mechanisms of rapid gene birth. Plant J 50(5):873–885. https://​
doi.​org/​10.​1111/j.​1365-​313x.​2007.​03097.x
Kumar S, Archak S, Tyagi RK, Kumar J, Vikas VK, Jacob SR et al
(2016) Evaluation of 19,460 wheat accessions conserved in the
Indian National Genebank to identify new sources of resistance
to rust and spot blotch diseases. PLoS ONE 11(12):e0167702.
https://​doi.​org/​10.​1371/​journ​al.​pone.​01677​02
Laskowski RA, Jabłońska J, Pravda L, Vařeková RS, Thornton JM
(2017) PDBsum: structural summaries of PDB entries. Protein
Sci 27(1):129–134. https://​doi.​org/​10.​1002/​pro.​3289
Lescot M (2002) PlantCARE, a database of plant cis-acting regu-
latory elements and a portal to tools for in silico analysis of
promoter sequences. Nucleic Acids Res 30(1):325–327. https://​
doi.​org/​10.​1093/​nar/​30.1.​325
Liu J, Wiberg DB, Zehnder AJ, Yang H (2007) Modeling the role
of irrigation in winter wheat yield, crop water productivity,
and production in China. Irrig Sci. https://​d oi.​o rg/​1 0.​1 007/​
s00271-​007-​0069-9
Liu B, Asseng S, Wang A, Wang S, Tang L, Cao W et al (2017)
Modelling the effects of post-heading heat stress on biomass
growth of winter wheat. Agric Meteorol 247:476–490. https://​
doi.​org/​10.​1016/j.​agrfo​r met.​2017.​08.​018
Lobell DB, Field CB (2007) Global scale climate–crop yield rela-
tionships and the impacts of recent warming. Environ Res Lett
2(1):014002. https://​doi.​org/​10.​1088/​1748-​9326/2/​1/​014002
Lobell DB, Sibley A, Ivan Ortiz-Monasterio J (2012) Extreme heat
effects on wheat senescence in India. Nat Clim Chang 2(3):186–
189. https://​doi.​org/​10.​1038/​nclim​ate13​56
Lovell SC, Davis IW, Arendall WB, de Bakker PIW, Word JM,
Prisant MG et al (2003) Structure validation by Cα geometry:
ϕ, ψ and Cβ deviation. Proteins 50(3):437–450. https://​doi.​org/​
10.​1002/​prot.​10286
Lush J (1949) Heritability of quantitative characters in farm animals.
Hereditas 35(S1):356–375. https://​doi.​org/​10.​1111/j.​1601-​5223.​
1949.​tb033​47.x
Ma X, Zhang J, Burgess P, Rossi S, Huang B (2018) Interactive
effects of melatonin and cytokinin on alleviating drought-
induced leaf senescence in creeping bentgrass (Agrostis stolonif-
era). Environ Exp Bot 145:1–11. https://​doi.​org/​10.​1016/j.​envex​
pbot.​2017.​10.​010
Mackinnon E, Stone SL (2022) The ubiquitin proteasome system
and nutrient stress response. Front Plant Sci. https://​doi.​org/​10.​
3389/​fpls.​2022.​867419
Marchler-Bauer A, Bo Y, Han L, He, J, Lanczycki CJ, Lu S et al
(2017) CDD/SPARCLE: functional classification of pro-
teins via subfamily domain architectures. Nucleic Acids Res
45(D1):D200–D203. https://​doi.​org/​10.​1093/​nar/​gkw11​29
Melo FV, Oliveira MM, Saibo NJM, Lourenço TF (2021) Modula-
tion of abiotic stress responses in rice by E3-ubiquitin ligases:
a promising way to develop stress-tolerant crops. Frontiers.
https://​doi.​org/​10.​3389/​fpls.​2021.​640193
Metzger MB, Hristova VA, Weissman AM (2012) HECT and RING
finger families of E3 ubiquitin ligases at a glance. J Cell Sci
125(3):531–537. https://​doi.​org/​10.​1242/​jcs.​091777
Metzger MB, Pruneda JN, Klevit RE, Weissman AM (2014) RING-
type E3 ligases: master manipulators of E2 ubiquitin-conjugat-
ing enzymes and ubiquitination. Biochim Biophys Acta (BBA)
1843(1):47–60. https://​doi.​org/​10.​1016/j.​bbamcr.​2013.​05.​026
Miricescu A, Goslin K, Graciet E (2018) Ubiquitylation in plants:
signaling hub for the integration of environmental signals. J
Exp Bot 69(19):4511–4527. https://​doi.​org/​10.​1093/​jxb/​ery165
Mirosavljević M, Mikić S, Župunski V, KondićŠpika A, Trkulja D,
Ottosen C et al (2021) Effects of high temperature during anthe-
sis and grain filling on physiological characteristics of winter
wheat cultivars. J Agron Crop Sci 207(5):823–832. https://​doi.​
org/​10.​1111/​jac.​12546
13
Plant Molecular Biology
Mukhopadhyay D, Riezman H (2007) Proteasome-independent
functions of ubiquitin in endocytosis and signaling. Science
315(5809):201–205. https://​doi.​org/​10.​1126/​scien​ce.​11270​85
Narayanan S, Tamura PJ, Roth MR, Prasad PV, Welti R (2016)
Wheat leaf lipids during heat stress: I. High day and night tem-
peratures result in major lipid alterations. Plant Cell Environ
39(4):787–803. https://​doi.​org/​10.​1111/​pce.​12649
Pathak NN, Nema DP (1985) Genetic advance in land-races of
wheat. Indian Journal Agricultural Sciences, New Delhi
Randall P, Moss H (1990) Some effects of temperature regime dur-
ing grain filling on wheat quality. Aust J Agric Res 41(4):603.
https://​doi.​org/​10.​1071/​ar990​0603
Reynolds MP, Singh RP, Ibrahim A, Ageeb OAA, Larque-Saavedra
A, Quick JS (1998) Evaluating physiological traits to comple-
ment empirical selection for wheat in warm environments.
Euphytica 100:85–94. https://​doi.​org/​10.​1023/A:​10183​55906​
553
Sahu S, Dhari R, Joshi AK (2005) Variability studies in wheat (Triti-
cum aestivum L.) under late sown condition. Indian J Genet Plant
Breed 65(04):309–310
Schmitz RJ, Grotewold E, Stam M (2022) Cis-regulatory sequences in
plants: their importance, discovery, and future challenges. Plant
Cell 34(2):718–741. https://​doi.​org/​10.​1093/​plcell/​koab2​81
Singh A, Singh PK, Sharma AK, Singh NK, Sonah H, Deshmukh R,
Sharma TR (2019) Understanding the role of the WRKY gene
family under stress conditions in pigeonpea (Cajanus cajan L.).
Plants 8(7):214. https://​doi.​org/​10.​3390/​plant​s8070​214
Stone SL (2014) The role of ubiquitin and the 26S proteasome in plant
abiotic stress signaling. Frontiers in Plant Science, 5. https://​doi.​
org/​10.​3389/​fpls.​2014.​00135
Stone SL (2019) Chapter three- role of the ubiquitin proteasome system
in plant response to abiotic stress. In: Galluzzi L (ed) International
review of cell and molecular biology. Academic Press, pp 65–110.
https://​doi.​org/​10.​1016/​bs.​ircmb.​2018.​05.​012
Stone SL, Callis J (2007) Ubiquitin ligases mediate growth and
development by promoting protein death. Curr Opin Plant Biol
10(6):624–632. https://​doi.​org/​10.​1016/j.​pbi.​2007.​07.​010
Tricker PJ, ElHabti A, Schmidt J, Fleury D (2018) The physiologi-
cal and genetic basis of combined drought and heat tolerance in
wheat. J Exp Bot 69(13):3195–3210. https://​doi.​org/​10.​1093/​jxb/​
ery081
Ullah S, Bramley H, Mahmood T, Trethowan R (2019) A strategy of
ideotype development for heat-tolerant wheat. J Agron Crop Sci
206(2):229–241. https://​doi.​org/​10.​1111/​jac.​12378
Vierstra RD (2003) The ubiquitin/26S proteasome pathway, the
complex last chapter in the life of many plant proteins. Trends
Plant Sci 8(3):135–142. https://​doi.​org/​10.​1016/​s1360-​1385(03)​
00014-1
Vierstra RD (2009) The ubiquitin–26S proteasome system at the nexus
of plant biology - Nature Reviews Molecular Cell Biology. Nature.
https://​doi.​org/​10.​1038/​nrm26​88
Vierstra RD (2012) The expanding universe of ubiquitin and ubiquitin-
like modifiers. Plant Physiol 160(1):2–14. https://d​ oi.o​ rg/1​ 0.1​ 104/​
pp.​112.​200667
Xiong R, Siegel D, Ross D (2013) The activation sequence of cellular
protein handling systems after proteasomal inhibition in dopamin-
ergic cells. Chem-Biol Interact 204(2):116–124. https://​doi.​org/​
10.​1016/j.​cbi.​2013.​04.​016
Yang D, Luo, Y, Ni Y, Yin Y, Yang W, Peng D, Cui Z, Wang Z (2014)
Effects of exogenous ABA application on postanthesis dry mat-
ter redistribution and grain starch accumulation of winter wheat
with different stay green characteristics. Crop J 2(2–3):144–153.
https://​doi.​org/​10.​1016/j.​cj.​2014.​02.​004
Yu X, Chen F, Chen Z, Wei P, Song X, Liu C, Liu T, Li X, Liu X
(2022) Genetic diversity and gene expression diversity shape the
adaptive pattern of the aquatic plant Batrachium bungei along an
Plant Molecular Biology
altitudinal gradient on the Qinghai-Tibet Plateau. Plant Mol Biol.
https://​doi.​org/​10.​1007/​s11103-​022-​01326-0
Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the
growth stages of cereals. Weed Res 14(6):415–421. https://​doi.​
org/​10.​1111/j.​1365-​3180.​1974.​tb010​84.x
Zampieri M, Ceglar A, Dentener F, Toreti A (2018) Understanding
and reproducing regional diversity of climate impacts on wheat
yields: current approaches, challenges and data driven limita-
tions. Environ Res Lett 13(2):021001. https://​doi.​org/​10.​1088/​
1748-​9326/​aaa00d
Zhao C, Liu B, Piao S, Wang X, Lobell DB, Huang Y et al (2017)
Temperature increase reduces global yields of major crops in four
independent estimates. Proc Natl Acad Sci USA 114(35):9326–
9331. https://​doi.​org/​10.​1073/​pnas.​17017​62114
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