Journal Journal

of Applied
Appl Journal of Applied Horticulture, 25(1): 3-9, 2023 Horticulture
DOI: https://doi.org/10.37855/jah.2023.v25i01.01 ISSN: 0972-1045

Proteomic landscape presents cues for vegetative to

reproductive transition in mango

Yashi Bajpai1,2, Sandeep Kumar1,2, M. Muthukumar1, S. Rajan1, Anju Bajpai1* and Mala
Trivedi2*
1
ICAR-Central Institute for Subtropical Horticulture, Lucknow-226101, India. 2Amity Institute of Biotechnology, Amity
University Uttar Pradesh, Lucknow Campus. *E-mail: [email protected]; [email protected]

Abstract
Proteome-based characterization of vegetative and flower bud formation was utilized to identify and differentiate protein species with
significant variable abundance during floral transition in mango cv. Dashehari using 2DE and corroborating the identified protein spots
using gene expression analysis. Total soluble proteins were phenol-extracted from the vegetative and floral flush of mango cv. Dashehari
and separated on 2D gels at pH 4-7. The protein spots with variable intensity were identified through SameSpots software. The protein
sequences of differentially accumulated spots were identified based on PI and MW using Citrus sinensis proteome isoelectric focusing
database. Furthermore, these protein sequences were used to conduct (tBLASTn) against Mangifera indica to predict the protein. Real
time gene expression was done to corroborate identified proteins. Total 301 spots were detected, out of which 16 were identified as
differentially expressed based on P value (P≤0.05) and a 2-fold change. These 16 protein spots were identified on the basis of in silico
comparative mapping protein against genome of mango and citrus, a close relative. They were classified into eight categories viz.,
transcriptional regulation, phenylpropanoid pathway and cell wall /cytoskeleton metabolism-related proteins, hormone signalling,
flowering time, signal-transduction, transport and protein synthesis associated to flowering. Five genes coding for shortlisted proteins
were used for validation of results using gene expression analysis. SAM (S adenosyl methionine synthase) was found to up-regulated
in floral flush, involved in the biosynthesis of polyamines has an association with flowering and stress responses. Furthermore, ARF
(Auxin Response Factor) and serine/threonine kinase gene members were also found to play a critical role in determining the floral
development process in mango, consistent with results obtained through 2DE. Protein species putatively involved in phenylpropanoid
pathway were also identified, showing the process of mango flowering from a new perspective beyond the conventional view. This
study of flowering-related proteomics provides an overview of the biological pathways and regulatory mechanisms associated with
the developmental physiology of flowering.
Key words: 2-DE, flowering, gene expression analysis, in silico comparative proteomics, mango, proteomics.

Introduction during flower bud differentiation in mango. Understanding the

systemic mechanism of floral induction in mangoes is complex.
Mango is one of the most important fruit globally and is a Factors like endogenous hormones, genetic makeup, soluble
commercially relevant crop. It is a terminal-bearing plant with carbohydrates, stress conditions as well as the age of the shoots
scanty knowledge of the variables influencing the transition from are crucial for successful flowering (Cho et al., 2017). In this
vegetative to reproductive mode. In general, mangoes go through context, effort has been made to identify important proteins
a juvenile stage after germination that lasts for several years and associated with floral transition in mango as there is a gap in
during which flowering does not occur and interactions with the knowledge regarding the role of regulatory pathways culminating
environment, flowering in the previous year, etc., are known to in floral transition under subtropical conditions. The cultivar
be responsible for phenomena like alternate bearing, thus making ‘Dashehari’ that dominates in Indian Gangetic plains exhibits the
it difficult for scientists to interpret the research data. typical alternate flowering behaviour, clearly defined on and off
years for flowering and fruiting. These characteristics make this
In accordance with a number of academic publications, scientists
cultivar a suitable model for studying the process of mango shoot
are currently using traditional genetic and molecular tools for the
maturation and flowering. The comparative analyses may aid in
discovery, identification, and cloning of floral-specific genes, a better understanding of the genetic and molecular regulatory
specifically the cloning of floral homeotic genes (Sung et al., mechanisms of flowering critical for phase transition (V-R).
2000; Weigel and Nilsson, 1995). With the rapid advancement
of proteome technology, the study of plant floral induction Materials and methods
mechanisms has been greatly accelerated (Kofler et al., 2022; Plant material and sampling time: The studies were conducted
Munoz-Fambuena et al., 2013). There is abundant literature on at the experimental farm of Central Institute for Subtropical
regulatory mechanisms of flowering in Arabidopsis, but there is Horticulture, Lucknow (latitude 26.90” N., longitude 80.76” E.)
scarcity regarding molecular regulatory mechanisms and different on a 15-year-old plants. The cultivar used was an alternate bearer,
expression levels of proteins, sugars and signalling molecules “Dashehari”, planted at 10 m spacing, with stabilized yield and
Journal of Applied Horticulture (www.horticultureresearch.net)
4 Proteomic landscape presents cues for vegetative to reproductive transition in mango

maintained under uniform cultural practices. Two types of shoots normalized spots and compared with the reference gel. The fold
(20 in no.) were tagged and their phenotypic fate was observed till difference and p-value were calculated using one-way ANOVA.
the bud burst stage: the mature shoots, called flowering bearing The threshold value for fold change was set at 2.0 for up and
shoots/floral flush and the new flush that continues to grow as down-regulation at P≤0.05. The protein spots were selected
vegetative flush (Fig. 1). The fully mature middle leaves of the based on spot intensity variations among vegetative and floral
shoots were selected and it was done from both types of the shoots flush. Further, these protein spots were identified based on PI and
in January 2021. During the experimental period, maximum MW characteristics, using Citrus sinensis proteome isoelectric
(20°C) and minimum (6.8°C) temperatures and relative humidity focusing database (http://isoelectricpointdb.org), as such database
(88.3-69.3) were recorded. The experiment was carried out with of Mangifera proteome is not available in the public domain. The
three biological replicates and each replication represented a protein sequences were further used to conduct protein-translated
single tree. Leaf samples were collected, rapidly frozen in liquid nucleotide (tBLASTn) against Mangifera indica (Taxid: 29780).
nitrogen, and stored at −80°C for further research.
Real-Time Quantitative PCR Validation: RNA isolation
Total protein extraction: Total proteins were extracted from and Real-Time were performed in floral and vegetative tissue
the vegetative and floral flush using the modified phenol method as reported previously by Bajpai et al. (2018). Gene-specific
(Carpentier et al., 2005). The protein concentration was calculated primer sets described were designed using the transcriptomic
using the Bradford protein method using bovine serum albumin data sets of vegetative and floral tissue of mango cv. Dashehari
as the reference standard (Bradford, 1976). submitted to the National Centre for Biotechnology information
Two-dimensional (2DE) polyacrylamide gel electrophoresis: (NCBI) (Accession numbers SRR11261955, SRR11261956). The
Two-dimensional gel electrophoresis was used to separate isolated housekeeping gene MiActin was taken as endogenous reference
crude protein in two dimensions using isoelectric focusing (IEF) gene to normalize the data (Luo et al., 2013). The fold change
gel in the first dimension and SDS PAGE in the second dimension in expression levels was calculated by ΔΔCT value as reported
as earlier standardized in our lab (Laxmi et al., 2022). previously Livak and Schmittgen (2001). All primers used for
qPCR are listed in (Table 1)
Image and data analysis: The gel was stained with Coomassie
blue, digitized using Epson Expression 11000XL Scanner (Fig. Fold Change = 2^(-ΔΔCt)
1), and analyzed using the SameSpots software (TotalLab Ltd, Results and discussion
UK). The spots were detected, matched, and normalized with
default parameters. Differentially expressed protein spots among 2DE reveals differential accumulation of flowering responsive
the vegetative and floral flush samples were ascertained using proteins in mango: The 2 D gels of mango cv Dashehari (leaves

Fig. 1. Type of shoot used for protein isolation and 2DE (A) Vegetative flush, (B) Floral flush, (C) Displaying protein spots in vegetative
flush, and (D) in floral flush.

Journal of Applied Horticulture (www.horticultureresearch.net)

 Proteomic landscape presents cues for vegetative to reproductive transition in mango 5

Fig. 2. Total soluble proteins resolved in 2D-gel. (A) Differentially expressed spots identified by Samespots software. (B) Close up view of
selected protein spots, in vegetative (left) and floral flush (right) (#586:CO5; #597: Trihelix; #726: AGL26 and #976: ARF) (C) Distribution
in different fu MYB53 and COMT)CO5, GA20ox, BCA1, ( Identified Key proteins Real time gene expression analysis database iefproteome
protein identification using Comparitive software Samespotsprotein spots using Identification of differentially expressed Total protein isolation
and 2DE Genetic Background GA3 IAA conditionsgrowth Optimal factorsgenetic Lack of Pathway regulated Vegetative -Up and GTP)
AP3, NAC, PPR, KEA3, F3H, SAM, TrihelixAGL 26, (Identified Key proteins Pathway regulated Flowering -UpGA3 IAA Hormonal status
Environmental factors Flowering Vegetative growth Mango Shoot Apical Meristem nctional categories based on comparative proteomics.

from vegetative and floral shoot) displayed 301 protein spots transcription factor was identified which is known to regulate
cumulatively. The proteins showed a broad distribution in the pI gene expression and flowering time by associating with the
(4.0 to 7.0) and mass (10 to 250 kDa), respectively. Based on spot histone demethylase JMJ14 (Ning et al., 2015). Another spot,
intensity variations among vegetative and floral flush samples #679, representing oxoglutarate-dependent dioxygenase proteins,
were quantified by SameSpots (version 5.1.012) software, where was identified. 2-oxoglutarate/Fe (II)-dependent dioxygenases
in 56 (18.60%) protein spots had 2-fold change (up or down) were related to early flowering in alfalfa (Ma et al., 2021). Both
between vegetative and floral flush, and only 16 spots (28.57%) of these proteins are found to be differentially up-regulated in the
that showed significant differences based on P≤0.05 and a 2-fold floral flush of mango cv. Dashehari. Protein spot #726 represents
change were used for further analysis (Fig. 2). Among them, ten the Agamous-like MADS-box protein. AGL is the crucial gene
were up-regulated, and six down-regulated. Identification of regulating floral transition and differentiation (Yadav et al.,
proteins based on in silico comparative synteny and annotations 2020). Furthermore, spot #586 was assigned as constans5, which
were selected by maximum query coverage and total score (Table encodes a member of the constans-like (COL) family. Earlier,
2). Some spot number codes for more than one protein, and we overexpression of ATCOL5 or ATCOL9 has been indicated to
selected the proteins based on their role in flowering as well as promote or repress flowering, respectively (Cheng and Wang,
those with the best score by (tBLASTn). The close-up view of 2005; Hassidim et al., 2009). The protein spot #525 represents
representative differentially expressed proteins is shown in Fig. 2. the Apetala3 that controls petal identity and floral meristem
patterning in Nigella damascene L. (Gonçalves et al., 2013) and
Classification of protein spots into different functional groups: determines the flowering fate of saffron (Wafai et al., 2018).
These proteins were categorized into different functional groups Protein spot #597 denotes the trihelix transcription factor which
such as transcriptional regulation, phenylpropanoid pathway and is essential for plant morphological developmental processes
cell wall /cytoskeleton metabolism-related proteins, hormone and regulating flowering time (Song et al., 2021). Likewise,
signalling, flowering time, signal-transduction, transport and protein spot #612 represents transcription factor MYB53, which
protein synthesis, and the percentage contribution pattern is is reported to regulate vegetative growth and is down-regulated
illustrated in pie chart (Fig. 2). Spot #488 representing NAC by SPL13 during reproductive development in Medicago
Table 1. The list of primers used for qRT-PCR
Spot no Protein name Forward primer Reverse primer
586 Constans 5 (CO5) 5’-AGCCGTTCTTCGACTAGCG-3’ 5’-GTTGTTGACACGCGCCACTG-3’
726 S-adenosylmethionine-dependent 5’-CATGGGGACGCTGGGCTTAC-3’ 5’-TCCACCTTGGTGGGGTCCTT-3’
methyltransferase (SAM)
597 Trihelix transcription factor (Trihelix) 5’-CACAGAATTGCCGCCAACAA-3’ 5’-ACCAGTCTTCCAATGCGACC-3’
726 Agamous-like MADS-box protein (AGL26) 5’-TTCTTTCGCTCTCTCGGCTT-3’ 5’-TGTGGCAGAGACACTCCATTC-3’
976 Auxin response factor-like protein (ARF) 5’-AACCACCCGCTGGATTTTGA-3’ 5’-AAGCCTGTGTAAGTGGTGGG-3’
537 Serine/threonine kinase gene (STK) 5’-TCAGCTGCAGTTGGCCTTGG-3’ 5’-GGCTGATCCGGACCTCAACG-3’
Reference Actin 5’GAGAGTTTTGATGTCCCTGCCATG-3’ 5’-CAACGTCGCATTTCATGATGGAGT-3’
gene

Journal of Applied Horticulture (www.horticultureresearch.net)

6 Proteomic landscape presents cues for vegetative to reproductive transition in mango

Table 2. Annotation results of differential spots based on synteny using in silico comparative proteomics
Spot Differential MW KD/ Matched Protein Best score Accession Pathway
No. expression PI (%)
488 Up 43/5.6 NAC domain-containing protein 71-like (NAC 87.50 XR_003066340.1 Flowering time
71-like)
679 Up 37/5.5 Probable 2-oxoglutarate-dependent dioxygenase 100 XM_006494416.3
(AOP2)
586 Down 40/5.7 Constans 5 (CO5) 73.10 MG677826.1

726 Up 35/5.7 Agamous-like MADS-box protein (AGL26) 100 XM_025096038.1

517 Up 42/5.9 Beta-tubulin gene (TUB2) 52.78 MK639361.1 Cell wall and cytoskeleton
metabolism
525 Up 42/5.5 AP-3 complex (AP-3) 100 XM_006472177.3 Transcription-al regulation

597 Up 40/4.4 Trihelix transcription factor (Trihelix) 80 XM_006475645.3

686 Down 36/6.5 ABC transporter I family member 6, (ABCA1) 99.69 XM_006485454.3

612 Down 39/6.1 Transcription factor MYB53 87.75 XM_024187525.1

679 Up 37/5.5 F-box proteins (FBPs) 58.08 XM_006451907.2

686 Down 36/6.5 Gibberellin 20-oxidase-like protein (GA20ox) 93.67 XM_006490166.2 Hormone signalling

976 Up 28/5.5 Auxin response factor-like protein (ARF) 90.3 AY255705.1

838 Up 32/5.0 Pentatricopeptide repeat-containing protein 99.66 XM_006420934.2 Protein metabolism
(PPR)
1261 Down 19/5.2 F-box/kelch-repeat protein 31.69 XM_031107395.1
(FBPs)
586 Down 40/5.7 Caffeic acid O-methyl 79.82 FJ645928.1 Phenyl- propanoid pathway
Transferase COMT)
976 Up 28/5.5 Flavanone 3-hydroxylase 2 (F3H) 32.37 KF956031.1
726 Up 35/5.7 S-adenosylmethionine-dependent 97.29 XM_024183454.1 Signal-transduction pathway
methyltransferase (SAM)
1261 Down 19/5.2 Receptor-like protein kinase (RLK) 82.56 XM_025094398.1

597 Up 40/4.4 GTP-binding protein (GTP) 84.1 XM_006466354.3

599 Down 39/5.7 NBS-LRR disease resistance protein (NBS- 42.35 HM446508.1
LRR)
537 Up 42/5.8 Serine/threonine kinase gene (STK) 73.7 AY693371.1

785 Down 33/5.2 Protein NDL1 100 XM_006432969.2

838 Up 32/5.0 Pentatricopeptide repeat- 99.66 XM_006420934.2

containing protein (PPR)

sativa L. (Gao et al., 2018). These MYB proteins participate transferase (COMT), a key enzyme in lignin biosynthesis, whose
in morphogenesis, control of specialized metabolism, circadian expression delayed flowering in rice (Li et al., 2009) and altered
clock control, response to phosphate starvation, and flower and flowering time in alfalfa (Sun et al., 2019). Two spots, #679
fruit development (Feller et al., 2011). One spot, #368, was and #1261, represented F-box protein and F-box/kelch-repeat
identified that represented K+ efflux antiporter 3. K+ transporters protein, respectively. F-box proteins are involved in regulating
were also shown to be essential for flower development via flowering, auxin signal transduction, floral organ formation and
maintaining K+ homeostasis in Arabidopsis and rice (Isayenkov leaf senescence (Kim et al., 2022). The latter spot was down-
et al., 2020). regulated, indicating that F-box/kelch-repeat proteins act as a
repressor during floral transition stages in mango. Spot #838,
Spot no. #517 represents the up-regulated Beta-tubulin (TUB2)
which codes for pentatricopeptide repeat-containing protein, was
gene, which plays a fundamental role in plant morphogenesis but
up-regulated, which was reported to be related to early flowering
also was related to flower development in flax (Gavazzi et al.,
(Saha et al., 2007), organelle biogenesis, and plant development
2017). Further, spot 686 represents the ABC transporter I family
(Emami and Kempken, 2019).
member 6, a class of ATP binding cassette proteins also known as
ABCG, which transport a variety of substrates necessary for plant Spot #976 and #686 represented Auxin response factor-like and
growth and reproduction (Zhao et al., 2016). Two Spots #586 and Gibberellin 20-oxidase-like proteins, respectively. Gibberellin 20
#976, were related to proteins of COMT and F3H, respectively. oxidase (GA20ox) is a key catalytic enzyme in the biosynthesis
The protein spot 976 represents Flavanone 3-hydroxylase 2, of GA, the concentration of which increases at the meristem
which is implicated in floral induction in saffron (Tu et al., immediately before floral induction and promotes flowering by
2016). Likewise, spot 586 denoted the Caffeic acid O-methyl up-regulating SOC1 expression (Zhou et al., 2019). Furthermore,
Journal of Applied Horticulture (www.horticultureresearch.net)
 Proteomic landscape presents cues for vegetative to reproductive transition in mango 7

another study showed a declining trend in gibberellic acid in

floral flush leaves compared to the vegetative flushes in different
mangoes, which explains the inhibitory potential of gibberellins
on mango flowering (Bajpai et al., 2021). Spot #726 corresponds
to the S-adenosyl methionine-dependent methyltransferase
(SAM) protein, which has been demonstrated to have a role in
flowering by several studies (Ding et al., 2015). Our protein spot
results agree with the hormonal theory of flowering, which states
that higher auxin levels with a dip in temperature and a decline
in gibberellin levels promote inductive flowering in subtropical
conditions in mango.
Functional categorization of protein spots from 2D gels placed
seven spots (726, 1261, 597, 599, 537, 785, 838) were categorized
under the signal transduction group, which are important for the
perception of environmental signals and transmission to cellular
machinery for activating different growth and development
responses in plants. Spot 726, 597, 537 and 838 were up-
regulated, while the remaining three spots (1261, 599 and 785)
were down-regulated. Likewise, the protein identified by spot Fig. 3. Validation of selected spots through qRT-PCR.
#597 represents an up-regulated GTP-binding protein that plays Protein NDL1, respectively. Pathogenesis-related proteins, e.g.
important roles in plant development, cell signalling, activation receptor-like proteins and toll-interleukin-like receptor (TIR)-
of DNA-binding protein of vernalization 1 (RTV1), resulting in nucleotide-binding site (NBS)-LRR class proteins and members
early flowering in Arabidopsis (Heo et al., 2012). Spot # 537 of the signalling receptor kinase family LRR proteins are related
denotes Serine/threonine-protein kinase was up-regulated, which to early flowering in alfalfa (Ma et al., 2021). The role of NDL1
has a role in stress-induced signalling, flower development and in the context with flowering is still not clear. Besides this, spot
regulating flowering time by modulating the photoperiod pathway #1261, which codes for the receptor-like protein kinase gene,
in Arabidopsis (Xie et al., 2022). Spot 599 and 785 display down- regulates cell morphogenesis, flowering time, and seed production
regulation and represent NBS-LRR disease resistance protein and (Gachomo et al., 2014).
Mango Shoot Apical Meristem

Genetic Background
Lack of Optimal
genetic growth
factors conditions
Environmental factors

Hormonal status
GA3 IAA GA3 IAA

Total protein isolation and 2DE

Identification of differentially expressed

protein spots using Samespots
software
Up-regulated Vegetative Up-regulated Flowering
Pathway Pathway

Identified Key proteins

(CO5, GA20ox, BCA1, Identified Key proteins
MYB53 and COMT) Comparitive protein identification using (AGL 26, Trihelix, SAM,
proteome ief database AP3, NAC, PPR, KEA3, F3H
and GTP)

Real time gene expression analysis

Vegetative growth Flowering

Fig. 4. Proteomics based model presents cues for vegetative to reproductive transition in mango

Journal of Applied Horticulture (www.horticultureresearch.net)

8 Proteomic landscape presents cues for vegetative to reproductive transition in mango

Validation by RT-PCR: In order to verify our 2DE results, six Dashehari using 2D gel electrophoresis. Among the identified
genes (CO5, SAM, Trihelix, AGL, ARF and STK) coding for 301 protein spots, 56 exhibited significant 2-fold changes, with 16
earlier mentioned proteins were selected for the RT-PCR analysis. spots selected for further analysis. These differentially expressed
These genes showed an apparent change between the vegetative proteins were categorized into functional groups, revealing their
and floral flush at the transcriptional level and were majorly up- roles in transcriptional regulation, signaling pathways, hormone
regulated in floral flush (Fig. 3). The expression profiles of all responses, and metabolic processes related to flowering. Notably,
of these genes at mRNA levels were consistent with those at the proteins such as NAC transcription factors, dioxygenases,
protein levels (Table 2). Since we know that CO5 is an activator MADS-box proteins, and transporters demonstrated significant
of flowering, yet here it is repressed post-transcriptionally in changes. Validation through RT-PCR confirmed differential
floral flush and up-regulated in vegetative, contributing to see expression of selected genes. This proteomic approach enhances
a new perspective to photoperiod sensitive flowering in mango our understanding of mango flowering dynamics and lays
except the conventional view. qRT-PCR showed that SAM gene groundwork for potential biomarkers for biennial bearing and
expression was comparatively high in floral flush as compared to floral regulation.
vegetative. SAM plays a central role in many cellular biochemistry
reactions, including flowering and fruit development (Ding et al., Acknowledgements
2015). Besides trihelix, a transcriptional activator was found to We wish to thank the Director, ICAR-Central Institute for
be up-regulated in floral flush with a log fold change of (0.87). Subtropical Horticulture, Lucknow, India, for providing guidance,
Several studies have demonstrated that a plant-specific trihelix necessary facilities and encouragement during the investigation.
transcription factor, one of a family previously known only as
regulators of light-controlled genes, causes early flowering (Song References
et al., 2021; Yang et al., 2020). We further found an up-regulation Bajpai, A., K. Khan, M. Muthukumar, S. Rajan and N.K. Singh, 2018.
of AGL26 gene in the floral flush (2.57) and its down-regulation Molecular analysis of anthocyanin biosynthesis pathway genes and
in the vegetative flush, which indicates towards the key role of their differential expression in mango peel. Genome, 61: 157-166.
AGL in regulating flowering initiation and development in mango. Bajpai, Y., M. Trivedi, M. Muthukumar and A. Bajpai, 2021. Novel
In a study, it was found that knocking down of AGL gene, leads insights into biochemical and hormonal factors regulating floral
to delayed flowering, whereas this gene’s constitutive expression transition in mango (Mangifera indica L.). Indian J. Biotechnol.,
caused rapid flowering in Arabidopsis (Zhou et al., 2021). 20: 54-64.
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation
AGL26, integrates multiple flowering pathway signals, and of microgram quantities of protein utilizing the principle of protein-
its expression levels eventually determine the exact flowering dye binding. Anal Biochem., 72: 248-254.
time (Van and Molenaar, 2017). Furthermore, the ARF (Auxin Carpentier, S.C., E. Witters, K. Laukens, P. Deckers, R. Swennen and B.
Response Factor) gene was also found to be up-regulated in floral Panis, 2005. Preparation of protein extracts from recalcitrant plant
flush (log fold change of 3.18), as compared with vegetative tissues: an evaluation of different methods for two-dimensional gel
(-0.08). Auxin Response Factors (ARFs) is reported to modulate electrophoresis analysis. Proteomics, 5: 2497-2507.
the expression of target genes by binding to auxin response Cheng, X.F. and Z.Y. Wang, 2005. Overexpression of COL9, a
elements (AuxREs) and influence the transcriptional activation CONSTANS‐LIKE gene, delays flowering by reducing expression
of CO and FT in Arabidopsis thaliana. Plant J., 43: 758-768.
of down-stream target genes, playing role in flower development
(Kumar et al., 2011). The expression trend of serine/threonine Cho, L.H., J. Yoon and G.An, 2017. The control of flowering time by
environmental factors. Plant J., 90: 708–719.
kinase during the floral development was found to be up-regulated
Ding, C., T. Chen, Y. Yang, S. Liu, K. Yan, X. Yue, H. Zhang, Y. Xiang,
in floral flush. Previous studies have also demonstrated that serine/
L. An and S. Chen, 2015. Molecular cloning and characterization
threonine kinase gene members may regulate circadian rhythms of an S-adenosyl methionine synthetase gene from Chorispora
and vacuolar H+-ATPase (Xie et al., 2022). bungeana. Gene, 572: 205-213.
There is a scarcity of data on the protein expression profile Emami, H. and F. Kempken, 2019. PRECOCIOUS 1 (POCO 1), a
mitochondrial pentatricopeptide repeat protein affects flowering time
during flower development in mango. This is the first proteomic in Arabidopsis thaliana. Plant J., 100: 265-278.
profiling study in mango cv. Dashehari during the period of
Feller, A., K. Machemer, E.L. Braun and E. Grotewold, 2011.
floral bud initiation and renders the possible development of Evolutionary and comparative analysis of MYB and bHLH plant
suitable biomarkers for biennial bearing in mango. We therefore transcription factors. Plant J., 66: 94-116.
performed proteome analysis of Mangifera indica cultivar Gachomo, E.W., L. Jno, Baptiste, T. Kefela, W.M. Saidel and S.O.
Dashehari vegetative and floral flush wherein putative proteins Kotchoni, 2014. The Arabidopsis CURVY1 (CVY1) gene encoding
playing an important role in mango biennial rhythm were a novel receptor-like protein kinase regulates cell morphogenesis,
identified through a proteomic-based approach and validated flowering time and seed production. BMC Plant Biol., 14: 1-9.
through real-time gene expression analysis (Fig. 4). Out of the Gao, R., M.Y. Gruber, L. Amyot and A. Hannoufa, 2018. SPL13 regulates
shoot branching and flowering time in Medicago sativa. Plant Mol.
observed differentially expressed proteins spots from the 2DE Biol., 96: 119-133.
data, the transcript abundance of six genes (CO5, SAM, Trihelix,
Gavazzi, F., G. Pigna, L. Braglia, S. Gianì, D. Breviario and L. Morello,
AGL26, ARF and STK), the key genes regulating floral transition 2017. Evolutionary characterization and transcript profiling of
and differentiation, were validated through qRT-PCR. The results β-tubulin genes in flax (Linum usitatissimum L.) during plant
of the study provide an overview of the important biological development. BMC Plant Biol., 17: 1-6.
processes occurring during flowering. Gonçalves, B., O. Nougué, F. Jabbour, C. Ridel, H. Morin, P. Laufs,
D. Manicacci and C. Damerval, 2013. An APETALA 3 homolog
In summary, the study unveils distinct protein accumulation controls both petal identity and floral meristem patterning in Nigella
patterns between vegetative and floral shoots of mango cv. damascena L. (Ranunculaceae). Plant J., 76: 223-235.
Journal of Applied Horticulture (www.horticultureresearch.net)
 Proteomic landscape presents cues for vegetative to reproductive transition in mango 9

Hassidim, M., Y. Harir, E. Yakir, I. Kron and R.M. Green, 2009. Saha, D., A.M. Prasad and R. Srinivasan, 2007. Pentatricopeptide repeat
Overexpression of CONSTANS-LIKE 5 can induce flowering in proteins and their emerging roles in plants. Plant Physiol. Biochem.,
short-day grown Arabidopsis. Planta, 230: 481-491. 45: 521-534.
Heo, J.B., S. Sung and S.M. Assmann, 2012. Ca2+ dependent GTPase, Song, J., W.Y. Shen, S. Shaheen, Y.Y. Li, Z.R. Liu, Z. Wang H.B. Pang
extra-large G protein 2 (XLG2), promotes activation of DNA-binding and Z. Ahmed, 2021. Genomewide identification and analysis of the
protein related to vernalization 1 (RTV1), leading to activation of trihelix transcription factors in sunflower. Biol. Plant., 65: 80-87.
floral integrator genes and early flowering in Arabidopsis. J. Biol. Sun, H., R. Long, F. Zhang, T. Zhang, J. Kang, Z. Wang, C. Cao, J.
Chem., 287: 8242-8253. Yu and Q. Yang, 2019. Proteomic analysis of shoot tips from two
Isayenkov, S.V., S.A Dabravolski, T. Pan and S. Shabala, 2020. alfalfa cultivars with different florescence. Plant Mol. Biol. Rep.,
Phylogenetic diversity and physiological roles of plant monovalent 37: 265-276.
cation/H+antiporters. Front. Plant Sci.,11: 573564. Sung, S.K., G.H. Yu, J. Nam, D.H. Jeong and G. An, 2000.
Kim, J.H., W.J. Jung, M.S. Kim, C.S. Ko, J.S. Yoon, M.J. Hong, H.J. Developmentally regulated expression of two MADS-box genes,
Shin and Y.W. Seo, 2022. Molecular characterization of wheat MdMADS3 and MdMADS4, in the morphogenesis of flower buds
floret development‐related F‐box protein (TaF‐box2): Possible and fruits in apple. Planta, 210: 519-528.
involvement in regulation of Arabidopsis flowering. Physiol. Plant., Tu, Y., F. Liu, D. Guo, L. Fan, Z. Zhu, Y. Xue, Y. Gao and M Guo,
174: e13677. 2016. Molecular characterization of flavanone 3-hydroxylase gene
Kofler, J., A. Milyaev, B. Würtz, J. Pfannstiel, H. Flachowsky and J.N. and flavonoid accumulation in two chemotyped safflower lines in
Wünsche, 2022. Proteomic differences in apple spur buds from response to methyl jasmonate stimulation. BMC Plant Biol., 16: 1-12.
high and non-cropping trees during floral initiation. J. Proteomics., Van, Dijk, A.D. and J. Molenaar, 2017. Floral pathway integrator gene
253: 104459. expression mediates gradual transmission of environmental and
Kumar, R., A.K Tyagi and A.K. Sharma, 2011. Genome-wide analysis of endogenous cues to flowering time. Peer J., 5: e3197.
auxin response factor (ARF) gene family from tomato and analysis Wafai, A.H., J.I. Mir, S. Bukhari, T.A. Mokhdomi, A. Amin, S.H. Wani
of their role in flower and fruit development. Mol. Genet. Genom., and R.A. Qadria, 2018. AP-3 gene expression study during flower
285: 245-260. development in saffron (Crocus sativus L.). Acta Hortic.,1200: 47-50.
Laxmi, A. Kamal., V. Kumar and A. Baja, 2022. Identification and Weigel, D. and O. Nilsson, 1995. A developmental switch sufficient for
characterization of salt-responsive proteins in mango (Mangifera flower initiation in diverse plants. Nature, 377: 495-500.
indica L.). J. Appl. Hortic., 24: 110-115.
Xie, K., Y. Wang, X. Bai, Z. Ye, C. Zhang, F. Sun, C. Zhang and Y. Xi,
Li, H., B. Handsaker, A. Wysoker, T. Fennell, J. Ruan, N. Homer, G. 2022. Overexpression of PvSTK1 gene from Switchgrass (Panicum
Marth, G. Abecasis and R. Durbin, 2009. Genome Project Data virgatum L.) affects flowering time and development of floral organ
Processing Subgroup. The Sequence Alignment/Map format and in transgenic Arabidopsis thaliana. Plant Physiol. Biochem., 178:
SAM tools. Bioinformatics, 25: 2078–2079. 93-104.
Livak, K.J. and TD. Schmittgen, 2001. Analysis of relative gene Yadav, A., P.K. Jayaswal, K. Venkat Raman, B. Singh, N.K. Singh and
expression data using real-time quantitative PCR and the 2− ΔΔCT K. Usha, 2020. Transcriptome analysis of flowering genes in mango
method. Methods, 25: 402-408. (Mangifera indica L.) in relation to floral malformation. J. Plant
Luo, C., XH He, H. Chen, Y. Hu and S.J. Ou, 2013. Molecular cloning Biochem. Biotechnol., 29: 193-212.
and expression analysis of four actin genes (MiACT) from mango. Yang, L., S. Qi, A. Touqeer, H. Li, X. Zhang, X. Liu and S. Wu, 2020.
Biologia Plantarum, 57: 238-244. SlGT11 controls floral organ patterning and floral determinacy in
Ma, D., B. Liu, L. Ge, Y. Weng, X. Cao, F. Liu, P. Mao and X. Ma, 2021. tomato. BMC Plant Biol., 20: 1-4.
Identification and characterization of regulatory pathways involved Zhao, G., J.W. D. Shi, Liang and Zhang, 2016. ATP binding cassette G
in early flowering in the new leaves of alfalfa (Medicago sativa L.) transporters and plant male reproduction. Plant Signal. Behav.,11:
by transcriptome analysis. BMC Plant Biol., 21: 1-14. e1136764.
Munoz-Fambuena, N., C. Mesejo, M. Agustí, S. Tarraga, D.J. Iglesias, Zhou, A., H. Sun, S. Dai, S. Feng, J. Zhang, S. Gong and J. Wang, 2019.
E. Primo-Millo and M.C. González-Mas, 2013. Proteomic analysis Identification of transcription factors involved in the regulation
of “Moncada” mandarin leaves with contrasting fruit load. Plant of flowering in Adonis amurensis through combined RNA-seq
Physiol. Biochem., 62: 95-106. transcriptomics and iTRAQ proteomics. Genes, 10: 305.
Ning, Y.Q., Z.Y. Ma, H.W. Huang, H. Mo, T.T. Zhao, L. Li, T. Cai, S. Zhou, F.Y., Q. Yu, Y. Zhang, Y.J. Han and C.C. Yao, 2021. Overexpression
Chen, L. Ma and X.J. He, 2015. Two novel NAC transcription factors of AGAMOUS-like gene PfAG5 promotes early flowering in
regulate gene expression and flowering time by associating with the Polypogon fugax. Funct. Plant Biol., 48: 793-801.
histone demethylase JMJ14. Nucleic Acids Res., 43: 1469-1484.
Received: December, 2022; Revised: December, 2022; Accepted: January, 2022

Journal of Applied Horticulture (www.horticultureresearch.net)