Genome-Wide Identification and Expression Pattern of the GRAS Gene Family in Pitaya (Selenicereus undatus L.)

Abstract

Simple Summary
The GRAS gene family plays a critical role in regulation of growth, defense, light and hormone responses. We identified 45 GRAS genes in the pitaya genome and categorized them into nine respective subfamilies: PAT1, SHR, LISCL, HAM, SCR, RGL, LAS, DELLA and SCL-3. Among these 45 HuGRAS family members, we reported nine candidate genes that played key roles in the growth and development of the pitaya plant.

Abstract
The GRAS gene family is one of the most important families of transcriptional factors that have diverse functions in plant growth and developmental processes including axillary meristem patterning, signal-transduction, cell maintenance, phytohormone and light signaling. Despite their importance, the function of GRAS genes in pitaya fruit (Selenicereus undatus L.) remains unknown. Here, 45 members of the HuGRAS gene family were identified in the pitaya genome, which was distributed on 11 chromosomes. All 45 members of HuGRAS were grouped into nine subfamilies using phylogenetic analysis with six other species: maize, rice, soybeans, tomatoes, Medicago truncatula and Arabidopsis. Among the 45 genes, 12 genes were selected from RNA-Seq data due to their higher expression in different plant tissues of pitaya. In order to verify the RNA-Seq data, these 12 HuGRAS genes were subjected for qRT-PCR validation. Nine HuGRAS genes exhibited higher relative expression in different tissues of the plant. These nine genes which were categorized into six subfamilies inlcuding DELLA (HuGRAS-1), SCL-3 (HuGRAS-7), PAT1 (HuGRAS-34, HuGRAS-35, HuGRAS-41), HAM (HuGRAS-37), SCR (HuGRAS-12) and LISCL (HuGRAS-18, HuGRAS-25) might regulate growth and development in the pitaya plant. The results of the present study provide valuable information to improve tropical pitaya through a molecular and conventional breeding program.

Full text

Citation: Zaman, Q.U.; Hussain,
M.A.; Khan, L.U.; Cui, J.-P.; Hui, L.;
Khan, D.; Lv, W.; Wang, H.-F.
Genome-Wide Identification and
Expression Pattern of the GRAS Gene
Family in Pitaya (Selenicereus undatus
L.). Biology 2023,12, 11. https://
doi.org/10.3390/biology12010011
Academic Editor: Valeria Terzi
Received: 30 November 2022
Revised: 15 December 2022
Accepted: 16 December 2022
Published: 21 December 2022
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biology
Article
Genome-Wide Identification and Expression Pattern of the
GRAS Gene Family in Pitaya (Selenicereus undatus L.)
Qamar U Zaman 1,2 , Muhammad Azhar Hussain 1,2, Latif Ullah Khan 1,2, Jian-Peng Cui 1,2, Liu Hui 1,2,
Darya Khan 1,2, Wei Lv 1,2 and Hua-Feng Wang 1,2,*
1Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute, Hainan University,
Sanya 572025, China
2College of Tropical Crops, Hainan University, Haikou 570228, China
*Correspondence: hfwang@hainanu.edu.cn
Simple Summary:
The GRAS gene family plays a critical role in regulation of growth, defense, light
and hormone responses. We identified 45 GRAS genes in the pitaya genome and categorized them
into nine respective subfamilies: PAT1, SHR, LISCL, HAM, SCR, RGL, LAS, DELLA and SCL-3.
Among these 45 HuGRAS family members, we reported nine candidate genes that played key roles
in the growth and development of the pitaya plant.
Abstract:
The GRAS gene family is one of the most important families of transcriptional factors that
have diverse functions in plant growth and developmental processes including axillary meristem
patterning, signal-transduction, cell maintenance, phytohormone and light signaling. Despite their
importance, the function of GRAS genes in pitaya fruit (Selenicereus undatus L.) remains unknown.
Here, 45 members of the HuGRAS gene family were identified in the pitaya genome, which was dis-
tributed on 11 chromosomes. All 45 members of HuGRAS were grouped into nine subfamilies using
phylogenetic analysis with six other species: maize, rice, soybeans, tomatoes, Medicago truncatula and
Arabidopsis. Among the 45 genes, 12 genes were selected from RNA-Seq data due to their higher
expression in different plant tissues of pitaya. In order to verify the RNA-Seq data, these 12 HuGRAS
genes were subjected for qRT-PCR validation. Nine HuGRAS genes exhibited higher relative expres-
sion in different tissues of the plant. These nine genes which were categorized into six subfamilies
inlcuding DELLA (HuGRAS-1), SCL-3 (HuGRAS-7), PAT1 (HuGRAS-34,HuGRAS-35,HuGRAS-41),
HAM (HuGRAS-37), SCR (HuGRAS-12) and LISCL (HuGRAS-18,HuGRAS-25) might regulate growth
and development in the pitaya plant. The results of the present study provide valuable information
to improve tropical pitaya through a molecular and conventional breeding program.
Keywords:
genome-wide analysis; GRAS gene family; growth and development; pitaya/dragon
fruit; S. undatus L.
1. Introduction
Pitaya fruit, also known as dragon fruit, belongs to the Cactaceae family, which com-
prises 127 genera and 1750 species [
1
]. Among these species, pitaya-Selenicereus undatus
(S. undatus) formerly known as Hylocereus undatus (2n = 2x = 22) is a diploid perennial climb-
ing plant that originated from rainforests in the tropical and subtropical regions of Mexico
and Colombia [
2
]. Pitaya fruit gained the attention of growers due to its attractive fuchsia
color, its delicious aroma and its ability to tolerate harsh environmental conditions [
1
,
3
].
It contains vitamin C and has high antioxidant properties linked to its phenolic and be-
tacyanin content [
4
]. Several transcription factors (TFs), including WRKY [
5
], MYB [
6
],
MADS-box [
7
], ARF [
8
], AP2/EREBP [
9
], HB [
10
], SBP [
11
], bZIP [
12
], APX [
13
] and the
GRAS family, are being explored to identify their specific roles in plants [
14
]. Significant
research has been conducted on the GRAS gene family in many crops, including Arabidop-
sis thaliana [
15
], Chinese cabbage [
16
], switchgrass [
17
]Medicago sativa [
18
], cassava [
19
],
Biology 2023,12, 11. https://doi.org/10.3390/biology12010011 https://www.mdpi.com/journal/biology

Biology 2023,12, 11 2 of 17
maize [
20
,
21
], rice [
22
], Melilotus albus [
23
], wheat [
24
], canola [
25
], foxtail millet [
26
] and
soybean [
27
], but rarely in tropical fruits such as litchi [
28
]. To improve the growth and
developmental process, it is important to report the GRAS gene family in pitaya fruit.
TFs are proteins that contain domains that bind to the promoter regions that transcribe
DNA into mRNA. The GRAS TF is named after the first reported TFs: gibberellic acid
insensitive (GAI) [
29
,
30
], REPRESSOR of GAI (RGA) [
31
] and SCARECROW (SCR) [
32
].
GRAS proteins consist of 360–850 amino acids, C-terminal homology and five carboxyl-
terminal motifs with the same sequence in the whole family [30,33,34]. The GRAS protein
can be divided into five peptide regions, and it carries highly conserved motifs in a specific
order: leucine heptad repeat I (LHRI), VHID, leucine heptad repeat II (LHRII), PFYRE and
SAW [
34
,
35
]. The GRAS gene family is involved in multiple phytohormone-signaling path-
ways and plays a diverse role in signal transduction [
35
,
36
], root patterning [
37
], meristem
formation, shoot development [35], axillary meristem patterning and cell maintenance.
The GRAS gene family is divided, based on its structure, into the following subfami-
lies: DELLA, HAIRY MERISTEM (HAM), LATERAL SUPRESSOR (LAS), Lilium longiflorum
SCARECROW-LIKE (LISCL), REPRESSOR OF GAI-LIKE (RGL), PHYTOCHROME A SIG-
NAL TRANSDUCTION 1 (PAT1), SCARECROW (SCR), SCARECROW-LIKE 3 (SCL3) and
SHORT ROOT (SHR) [
35
,
38
–
40
]. GRAS subfamilies perform transcriptional regulation and
are involved in specific functions, as the DELLA subfamily negatively interacts with the
gibberellic-acid (GA) and light-signaling pathways [
41
], HAM with shoot-stem-cell initiation
and proliferation [
42
] and LAS with axillary meristems [
43
,
44
]. The LISCL/TGA complex
responds to defense and stress tolerance, PAT1 interacts with phytochrome-A signal trans-
duction [
45
] and SCR interacts with radial patterning in roots and shoots [
46
]. SCL3 acts as
an attenuator of DELLA proteins and represses their expression antagonistically [
47
], and
SHR interacts with endodermis specification and root patterning [
37
]. Through genome-
wide analysis, different GRAS genes have been predicted in different crop species: 48 in
Chinese cabbage [
16
], 144 in switchgrass [
17
], 87 in canola (Brassica napus) [
25
], 59 in Med-
icago truncatula [
48
], 52 in quinoa [
49
], 117 in soybeans (Glycine max L.) [
21
], 150 in cotton
(Gossypium hirsutum) [
50
], 62 in barley (Hordeum vulgare) [
51
], 37 in bottle gourds [
52
], 50 in
sweet oranges [
53
], 48 in litchi (Litchi chinesis Sonn) [
28
], 57 in rice (Oryza sativa L.) and maize
(Zea mays L.) [
30
], 55 in Melilotus albus [
23
], 50 in pepper (Capsicum annuum L.) [
54
] and 32 in
Arabidopsis thaliana [55].
In this study, we performed a comprehensive genome-wide analysis of the GRAS gene
family and identified 45 GRAS members in the pitaya (S. undatus L.) genome, mapped to
11 chromosomes (chrs). The GRAS genes were identified, and phylogenetic relationships were
established with the previously reported GRAS proteins of maize (Zea mays L.), soybeans
(Glycine max L.), Mediacgo trunctula, rice (Oryza sativa L.), Arabidopsis thaliana and tomatoes
(Solanum lycopersicum). We identified the locations of 45 genes on the chromosome and
selected 12 genes to check their expression patterns in different tissues of the pitaya plant.
Among them, HuGRAS-1,HuGRAS-6,HuGRAS-12,HuGRAS-18,HuGRAS-25,HuGRAS-34,
HuGRAS-35,HuGRAS-37 and HuGRAS-41 were found to be important genes that exert their
potential functions in the growth and development of the pitaya (S. undatus L.) plant.
2. Materials and Methods
2.1. Retrieval of GRAS Family Members in Pitaya
The genome of the pitaya plant (S. undatus L.) was downloaded from the pitaya genome
database, http://www.pitayagenomic.com/ (accessed on 1 September 2022) [
56
]. All pre-
viously published information about GRAS proteins was retrieved from the NCBI website,
https://www.ncbi.nlm.nih.gov/protein (accessed on 24 September 2022), and Phytozome,
https://phytozome-next.jgi.doe.gov/ (accessed on 2 October 2022) [
57
]. The InterPro tool,
https://www.ebi.ac.uk/interpro/ (accessed on 29 November 2022), was used to find the
domains of the GRAS proteins. The characterized protein sequences of corn (Zea mays L.) [
30
],
soybeans (Glycine max L.) [
21
], Mediacgo truncatula [
48
], rice (Oryza sativa L.) [
55
], Arabidopsis
thaliana [
46
] and tomatoes (Solanum lycopersicum) [
58
] were obtained from previous studies.

Biology 2023,12, 11 3 of 17
GRAS-protein physical and chemical properties, including each protein’s molecular weight,
isoelectric point and grand average of hydropathicity (GRAVY), were computed using the
Expasy ProtParam Tool, https://web.expasy.org/protparam/ (accessed on 9 October 2022).
2.2. Domain Analysis of HuGRAS Proteins
All 45 HuGRAS protein sequences were subjected to finding of conserved domains
using the NCBI conserved domain tool, https://www.ncbi.nlm.nih.gov/Structure/cdd/
wrpsb.cgi (accessed on 13 October 2022), against Pfamv34.0-19178pSSMs. The retrieved
data was used to draw the structure of the GRAS domain using TBTools [
59
]. A motif finder,
https://www.genome.jp/tools/motif/ (accessed on 17 October 2022), was also used to
compute the concerned motifs.
2.3. Phylogenetic Analysis of HuGRAS Family
All GRAS protein sequences of Arabidopsis (A. thaliana araport11), Medicago trun-
catula (Medicago truncatula Mt4.0v1—barrel medic), soybeans (Glycine max Wm82.a4.v1),
rice (Oryza sativa—v7.0), tomatoes (Solanum lycopersicum—ITAG4.0) and corn (Zea mays—
Refgen_V4) were downloaded from Phytozome-13 (https://phytozome-next.jgi.doe.gov
(accessed on 13 October 2022)) [
57
], and pitaya (S. undatus—Guanhuabai) protein sequences
were retrieved from the “pitaya-genome-website” [
56
]. All 380 protein sequences were used
to perform alignment through molecular evolutionary genetic analysis (MEGA-11) [
60
].
The aligned protein sequences were employed for phylogenetic analysis with the maximum
likelihood tree test, which used 1000 bootstrap replicates. Finally, the tree was visualized
using iTOL software [61].
2.4. HuGRAS Genes Distribution on Pitaya Chromosomes
All 45 GRAS genes’ data and genomic DNA were obtained from the pitaya genome
database, http://www.pitayagenomic.com/ (accessed on 1 September 2022) [
56
]. Chromo-
some length was calculated using TBTools “FASTA stats” [
59
]. Then, a chr ideogram was cre-
ated using the PhenoGram plot tool, http://visualization.ritchielab.org/phenograms/plot
(accessed on 1 September 2022). The output image thereof was used to mention all HuGRAS
genes on the chromosome.
2.5. Pattern and Distribution of Conserved Motifs
The MEME suite program was used to analyze the 45 HuGRAS genes and identify
conserved motifs. The default parameters were used to identify the maximum 10 motifs at
https://meme-suite.org/meme/tools/meme (accessed on 13 October 2022).
2.6. Expression Analysis of HuGRAS Genes
The expression pattern of the HuGRAS gene family was found via the pitaya genome
database (http://www.pitayagenomic.com/ (accessed on 17 October 2022)) [
56
]. All
45 HuGRAS genes’ expression levels were determined using different tissues of the plants,
including four stages of flower buds (FB1, FB2, FB3, FB4), five stages of flower (F1, F2, F3,
F4, F5), three stages of pericarp (PeriC-45d, PeriC-65d, PeriC-85d) and three stages of fruit
pulp (Pulp-29d, Pulp-35d, Pulp-49d).
2.7. Network Analysis of HuGRAS Proteins
All 45 HuGRAS genes were subjected to collection of their interactions with other
genes using the pitaya genome website, http://www.pitayagenomic.com/coexpression
(accessed on 30 October 2022) [
56
]. The Cytoscape tool was used to build the network using
information from all of the identified and interacting genes [62].

Biology 2023,12, 11 4 of 17
2.8. Cis-Acting Element Analysis in HuGRAS Promoter Sequences
Promoter sequences were retrieved from the pitaya genome file using TBTools (2000 bp
upstream of the start codon) [
59
]. The PlantCARE database was used to retrieve cisacting
regulatory elements [63].
2.9. Plant Materials
The “Shuangse Dahong” pitaya variety was used in this experiment, and flower buds
were collected from the germplasm resource of Hainan-Shengda Modern Agriculture De-
velopment Company, Qionghai, Hainan, China. All plants were grown in field conditions.
2.10. RNA Isolation and Real-Time Quantitative PCR Expression Analysis
Utilizing the RNAprep Pure Plant Kit, total RNA was extracted (TIANGEN, Beijing,
China). The plant material used for RNA extraction included stems (one-month-old stems,
one-year-old stems and two-year-old stems, designated S1Ms, S1Ys and S2Ys, respectively),
flower buds (FBs), pericarp (PeriC) and pulp. A NanoDrop 2000C spectrophotometer was
used to measure the concentration of the samples (Thermo Fisher Scientific, Waltham, MA,
USA). DNase I was used to remove genomic DNA from a total of 1 g of RNA from each
sample before being utilized as a template for reverse transcription to create the desired
amount of cDNA (QuantiTect Reverse Transcription Kit; Qiagen, Shanghai, China). The
RNA sample for each qRT-PCR was standardized using the actin-gene-expression level in
S. undatus L. Three biological and three technical replications of each sample were used
in the qPCR, with ACTIN serving as the internal control. The SYBER Green Master Mix
(Novogene, Shanghai, China) was used, along with the LightCycler 480 real-time PCR
system (Applied Biosystem, St. Louis, MO, USA). qRT-PCR results were analyzed using
the double-delta CT method [64,65].
3. Results
3.1. Genome-Wide Identification of the GRAS Family in Pitaya
Through genome-wide analysis, 45 candidate genes were retrieved from the pitaya
genome, and these genes were designated HuGRAS-1 to HuGRAS-45. Basic physical and
chemical properties of the HuGRAS genes, including each gene’s chromosome number,
position on the chromosome, CDS length, protein length, protein molecular weight, iso-
electric point (pI) and GRAVY, are summarized in Table 1. Protein length and molecular
weight varied greatly, ranging from 97 (HU08G00229.1) to 809 AA (HU01G00472.1), and
molecular weight ranged from 15–95 kDa. All 45 pitaya HuGRAS proteins were predicted
to be hydrophilic because their representative GRAVY values were less than 0, ranging
from
−
0.006 (HU06G00376.1) to
−
0.436 (HU02G01570.1). All HuGRAS proteins comprised
varying degrees of pI values, ranging from 5.4 (HU10G00709.1) to 9.5 (HU08G00229.1), with
an average value of 7.2.
Table 1. Physical and chemical properties of GRAS genes in pitaya (S. undatus L.).
Transcript ID Renamed ID Chr. No. Start–End
Position CDS (bp) Protein
Length (AA)
Protein Mol.
Weight (kDa) pI GRAVY
HU10G00709.1 HuGRAS-1 10 8,621,296-
8,623,893 1755 584 40.14 5.44 −0.055
HU11G00778.1 HuGRAS-2 11 78,577,603-
78,578,866 1176 391 39.90 6.63 0.105
HU03G02797.1 HuGRAS-3 3
132,126,059-
132,127,531 1473 490 40.39 6.23 −0.027
HU04G00148.1 HuGRAS-4 4
1,819,377-
1,821,914 2055 684 42.26 5.89 −0.221

Biology 2023,12, 11 5 of 17
Table 1. Cont.
Transcript ID Renamed ID Chr. No. Start–End
Position CDS (bp) Protein
Length (AA)
Protein Mol.
Weight (kDa) pI GRAVY
HU08G00367.1 HuGRAS-5 8
14,545,649-
14,547,799 1350 449 44.77 6.09 −0.13
HU01G01391.1 HuGRAS-6 1
95,463,256-
95,466,959 1623 540 41.20 6.9 −0.101
HU08G01232.1 HuGRAS-7 8
93,024,447-
93,026,894 1392 463 46.25 6.31 −0.086
HU08G00284.1 HuGRAS-8 8
9,185,291-
9,193,945 1533 510 41.50 5.91 −0.067
HU08G00014.1 HuGRAS-9 8
524,930-
530,436 1779 592 41.79 8.7 −0.234
HU05G01267.1 HuGRAS-10 5
102,083,025-
102,085,852 1332 443 46.94 5.99 −0.12
HU01G01850.1 HuGRAS-11 1
126,187,756-
126,191,336 1743 580 41.77 6.3 −0.167
HU01G00472.1 HuGRAS-12 1
6,212,129-
6,216,403 2430 809 38.49 5.76 −0.016
HU02G03005.1 HuGRAS-13 2
138,909,434-
138,911,167 1227 408 39.46 6.44 −0.017
HU03G01737.1 HuGRAS-14 3
25,880,385-
25,881,812 1428 475 46.22 6.57 −0.19
HU11G01125.1 HuGRAS-15 11
87,396,169-
87,400,880 1704 567 41.58 7.1 −0.142
HU11G01134.1 HuGRAS-16 11
87,665,022-
87,669,731 1704 567 41.58 7.1 −0.142
HU01G00833.1 HuGRAS-17 1
16,765,953-
16,767,335 1383 460 34.07 6.36 −0.213
HU02G01569.1 HuGRAS-18 2
22,485,375-
22,488,601 2283 760 42.05 8.6 −0.24
HU06G00568.1 HuGRAS-19 6
6,285,841-
6,289,986 2313 770 42.09 9.09 −0.239
HU05G01597.1 HuGRAS-20 5
117,157,311-
117,159,569 2259 752 42.17 9.46 −0.236
HU07G02248.1 HuGRAS-21 7
112,127,788-
112,125,521 2268 755 42.85 9.24 −0.295
HU07G02249.1 HuGRAS-22 7
112,130,817-
112,133,676 2265 754 42.53 8.96 −0.298
HU06G00029.1 HuGRAS-23 6
372,597-
376,546 1698 565 35.31 6.3 −0.092
HU02G01571.1 HuGRAS-24 2
22,556,900-
22,559,249 2001 666 42.22 8.95 −0.18
HU07G02246.1 HuGRAS-25 7
112,113,563-
112,116,265 1404 467 42.53 9.15 −0.333
HU07G00272.1 HuGRAS-26 7
2,834,842-
2,836,221 1380 459 42.87 5.5 −0.18
HU11G01529.1 HuGRAS-27 11
92,622,998-
92,624,392 1395 464 43.24 5.73 −0.171
HU06G00358.1 HuGRAS-28 6
3,831,873-
3,833,640 1458 485 42.91 5.98 −0.24
HU02G01572.1 HuGRAS-29 2
22,590,054-
22,592,716 2061 686 42.18 9.43 −0.274
HU02G01570.1 HuGRAS-30 2
22,492,850-
22,495,255 2406 801 42.68 8.1 −0.436
HU02G01573.1 HuGRAS-31 2
22,646,432-
22,648,477 2046 681 42.35 9.17 −0.367
HU05G00466.1 HuGRAS-32 5
8,309,337-
8,311,578 1770 589 39.79 6.86 0.143
HU04G00047.1 HuGRAS-33 4
727,912-
731,196 2310 769 39.78 6.33 0.157
HU06G02537.1 HuGRAS-34 6
123,795,575-
123,798,959 2289 762 40.48 7.86 −0.138
HU08G02295.1 HuGRAS-35 8
106,955,054-
106,955,638 585 194 21.76 8.89 −0.617

Biology 2023,12, 11 6 of 17
Table 1. Cont.
Transcript ID Renamed ID Chr. No. Start–End
Position CDS (bp) Protein
Length (AA)
Protein Mol.
Weight (kDa) pI GRAVY
HU05G01983.1 HuGRAS-36 5
124,288,256-
124,289,817 1455 484 44.22 5.84 −0.235
HU04G00048.1 HuGRAS-37 4
739,383-
742,094 2016 671 41.56 6.59 0.059
HU06G00376.1 HuGRAS-38 6
4,097,194-
4,098,474 1281 426 43.01 5.1 0.006
HU08G00019.1 HuGRAS-39 8
594,594-
594,875 726 241 16.98 6.93 −0.151
HU06G00283.1 HuGRAS-40 6
594,594-
602,896 1371 456 40.78 8.54 0.127
HU08G02296.1 HuGRAS-41 8
106,955,745-
106,956,677 933 310 15.39 7.01 0.115
HU08G00230.1 HuGRAS-42 8
6,804,389-
6,812,672 723 240 16.67 7.95 −0.181
HU02G03154.1 HuGRAS-43 2
141,447,089-
141,448,403 1185 394 37.68 5.87 0.42
HU05G02245.1 HuGRAS-44 5
126,702,616-
126,704,494 1617 538 40.33 8.52 0.201
HU08G00229.1 HuGRAS-45 8
6,804,079-
6,804,372 294 97 95.15 9.56 −0.032
Furthermore, domain-based analysis was carried out for all 45 HuGRAS proteins using
an NCBI domain search, and the retrieved data and TBTools were further used to draw the
structure of the domain. This domain-based analysis confirmed the presence of the GRAS
family on 45 selected protein sequences (Figure 1).
Biology2023,12,xFORPEERREVIEW7of20



Figure1.GRAS‐familyproteindomains.All45HuGRASsequences(HuGRAS‐1toHuGRAS‐45)
containedGRASdomains.
3.2.PhylogeneticAnalysisoftheGRASGeneFamily
Toconstructaphylogenetictree,proteinsequencesfromthecharacterizedspecies
wereretrievedfrompreviousstudies,includingthoseonArabidopsis,Medicagotrun‐
catula,tomatoes,rice,soybeansandmaize.CharacterizedGRASproteinsequencesfrom
sixspeciesandthePhytozomewebsitewereusedtoretrieveallGRASproteinsfromtheir
respectivegenomes.Collectively,380proteinsequenceswereusedtodrawaphylogenetic
tree,includingthe45HuGRASproteinsequencesfromthepitayagenomeand335protein
sequencesfromtheothersixspecies(SupplementaryFileS1).Intheresultingphyloge‐
netictree,theGRASgenesweredividedintoninesubfamilies:PAT1,SHR,LISCL,HAM,
SCR,RGL,LAS,DELLAandSCL3(Figure2).All45HuGRASproteinsweregroupedas
follows:twelveinPAT1;teninLISCL;fiveinHAM;foureachinSHR,SCL3andSCR;
threeinDELLA;twoinLAS;andoneinRGL.ThePAT1subfamilycontained12typesof
HuGRASproteinandwasthelargestsubfamilyofGRASprotein,whileRGLhadonlyone
typeofHuGRASproteinandwasoneofthesmallestsubfamiliesofGRASprotein.
Figure 1.
GRAS-family protein domains. All 45 HuGRAS sequences (HuGRAS-1 to HuGRAS-45)
contained GRAS domains.

Biology 2023,12, 11 7 of 17
3.2. Phylogenetic Analysis of the GRAS Gene Family
To construct a phylogenetic tree, protein sequences from the characterized species
were retrieved from previous studies, including those on Arabidopsis, Medicago truncatula,
tomatoes, rice, soybeans and maize. Characterized GRAS protein sequences from six
species and the Phytozome website were used to retrieve all GRAS proteins from their
respective genomes. Collectively, 380 protein sequences were used to draw a phylogenetic
tree, including the 45 HuGRAS protein sequences from the pitaya genome and 335 protein
sequences from the other six species (Supplementary File S1). In the resulting phylogenetic
tree, the GRAS genes were divided into nine subfamilies: PAT1, SHR, LISCL, HAM, SCR,
RGL, LAS, DELLA and SCL3 (Figure 2). All 45 HuGRAS proteins were grouped as follows:
twelve in PAT1; ten in LISCL; five in HAM; four each in SHR, SCL3 and SCR; three in
DELLA; two in LAS; and one in RGL. The PAT1 subfamily contained 12 types of HuGRAS
protein and was the largest subfamily of GRAS protein, while RGL had only one type of
HuGRAS protein and was one of the smallest subfamilies of GRAS protein.
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Figure2.Characterizedsequencesofsixspecies(maize,soybeans,Arabidopsis,Medicagotruncatula,
tomatoesandrice)wereusedtodrawthisphylogenetictreewiththepitayaGRASgenes.TheGRAS
proteinsweredividedintoninesubfamilies,exhibitedwithdifferentcolors:PAT1(red),SHR(navy
blue),LISCL(lime),HAM(lightorange/wheatcolor),SCR(violet),RGL(peagreen),LAS(teal),
DELLA(magentapink)andSCL3(gray).
3.3.HuGRAS‐ProteinSequenceAlignmentsandConservedMotifs
Aconserved‐motifanalysisofeachGRASproteinwascarriedoutusingtheMEME
tool.Tenconservedmotifswereidentified.TheCterminalregionsoftheHuGRASpro‐
teinscontainedhighlyconserveddomains.Motif2,motif6andmotif7werefoundin
almostalloftheHUGRASproteins.However,motif5wasnotfoundinHuGRAS‐35,
HuGRAS‐39,HuGRAS‐42orHuGRAS‐45(Figure3).MostoftheGRASproteinscarried
similarmotifswithinthegroup,withveryfewmotifdifferences.Thesefindingsalso
helpedustounderstandthecloseevolutionaryrelationshipsofthesameproteingroup.
Theknown‐motifoftheamino‐acid‐sequenceisexhibitedinFigureS1.
Figure 2.
Characterized sequences of six species (maize, soybeans, Arabidopsis, Medicago truncatula,
tomatoes and rice) were used to draw this phylogenetic tree with the pitaya GRAS genes. The GRAS
proteins were divided into nine subfamilies, exhibited with different colors: PAT1 (red), SHR (navy
blue), LISCL (lime), HAM (light orange/wheat color), SCR (violet), RGL (pea green), LAS (teal),
DELLA (magenta pink) and SCL3 (gray).

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3.3. HuGRAS-Protein Sequence Alignments and Conserved Motifs
A conserved-motif analysis of each GRAS protein was carried out using the MEME
tool. Ten conserved motifs were identified. The C terminal regions of the HuGRAS proteins
contained highly conserved domains. Motif 2, motif 6 and motif 7 were found in almost
all of the HUGRAS proteins. However, motif 5 was not found in HuGRAS-35,
HuGRAS-39
,
HuGRAS-42 or HuGRAS-45 (Figure 3). Most of the GRAS proteins carried similar motifs
within the group, with very few motif differences. These findings also helped us to
understand the close evolutionary relationships of the same protein group. The known-
motif of the amino-acid-sequence is exhibited in Figure S1.
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Figure3.DistributionofputativemotifsineachHuGRASproteinsequence.(A)Therectangular
phylogenetictreeof45HuGRASproteinswasconstructedusingMEGA‐11softwarebasedonthe
maximumlikelihoodmethod,withabootstrapvalueof1000replicates.(B)Conservedmotifsof
pitaya,namedHuGRAS‐1toHuGRAS‐45,thatwerepredictedusingtheMEMEprogramandplot‐
tedinTBToolssoftware.Motif1tomotif10areshownindifferentlycoloredboxes.
3.4.GeneStructureandDistributionofHuGRASGenesonChromosomes
Tofindthegenestructures,theintronandexonstructuresofalloftheHuGRASgenes
werealigned(FigureS2).Amongall45HuGRASgenes,mostoftheHuGRASgenese‐
quencesshowedtwosequencesofintronsandonesequenceofexons.Themajorityofthe
HuGRASgeneshadasimilarpatternofexons,indicatingthephylogenyandevolutionof
theirgenefamily,exceptforHuGRAS‐8andHuGRAS‐42.
TheHuGRASgeneswerephysicallylocatedon11chrsinthepitayagenome.All
GRASgenesweremappedonpitayachrsbasedontheinformationavailableatthepitaya
genomewebsite,http://www.pitayagenomic.com/(accessedon1September2022).The
chrlengthandpositionofeachgeneofthepitayagenomeispresentedinSupplementary
FileS2.Forty‐fiveHuGRASgeneswereunevenlydistributedon11chrs.Mostofthe
HuGRASgeneswerefoundonchr02andchr08.Chr10hadoneGRASgenebutchr09
hadnoHuGRASgenes(Figure4).
Figure 3.
Distribution of putative motifs in each HuGRAS protein sequence. (
A
) The rectangular
phylogenetic tree of 45 HuGRAS proteins was constructed using MEGA-11 software based on the
maximum likelihood method, with a bootstrap value of 1000 replicates. (
B
) Conserved motifs of
pitaya, named HuGRAS-1 to HuGRAS-45, that were predicted using the MEME program and plotted
in TBTools software. Motif 1 to motif 10 are shown in differently colored boxes.
3.4. Gene Structure and Distribution of HuGRAS Genes on Chromosomes
To find the gene structures, the intron and exon structures of all of the HuGRAS
genes were aligned (Figure S2). Among all 45 HuGRAS genes, most of the HuGRAS gene
sequences showed two sequences of introns and one sequence of exons. The majority of
the HuGRAS genes had a similar pattern of exons, indicating the phylogeny and evolution
of their gene family, except for HuGRAS-8 and HuGRAS-42.
The HuGRAS genes were physically located on 11 chrs in the pitaya genome. All GRAS
genes were mapped on pitaya chrs based on the information available at the pitaya genome
website, http://www.pitayagenomic.com/ (accessed on 1 September 2022). The chr length
and position of each gene of the pitaya genome is presented in Supplementary File S2.
Forty-five HuGRAS genes were unevenly distributed on 11 chrs. Most of the HuGRAS

Biology 2023,12, 11 9 of 17
genes were found on chr 02 and chr 08. Chr 10 had one GRAS gene but chr 09 had no
HuGRAS genes (Figure 4).
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Figure4.Distributionof45HuGRASgeneson11pitaya(S.undatusL.)chromosomes.Genenames
arementionedinblack.HuGRASgenesaredividedintoninegroupsonthebasisoftheirdomain
structures.
3.5.ExpressionAnalysisofHuGRASGenesinDifferentTissuesofPitaya
GRASTFsandGRASsubfamilymembers,includingDELLA,HAM,LAS,LISCL,
PAT1,SCR,SCL3,SHRandRGL,playimportantrolesinplantgrowthanddevelopment,
axillarymeristemformation,rootradialpatterning,cellmaintenanceandproliferation,
defenseresponseandstresstolerance.Thegenesineachtissueplaycentralrolesinpitaya
development.TheexpressionofGRASgenesinthepitayaplantcomprises15tissues,in‐
cludingflowerbuds(fourstages—FB1toFB4),flowers(fivestages—F1toF5),pericarp
(threestages—45days,65days,85days)andthepulpofthefruit(threestages—29days,
35days,49days).Ofthe45HuGRASgenes,mostwerenotexpressed(Figure5).We
choose12genesthatshowedsignificantdifferentialexpressioninalltissues:HuGRAS‐1,
HuGRAS‐6,HuGRAS‐7,HuGRAS‐12,HuGRAS‐18,HuGRAS‐21,HuGRAS‐25,HuGRAS‐29,
HuGRAS‐34,HuGRAS‐35,HuGRAS‐37andHuGRAS‐41.

Figure 4.
Distribution of 45 HuGRAS genes on 11 pitaya (S. undatus L.) chromosomes. Gene names are
mentioned in black. HuGRAS genes are divided into nine groups on the basis of their domain structures.
3.5. Expression Analysis of HuGRAS Genes in Different Tissues of Pitaya
GRAS TFs and GRAS subfamily members, including DELLA, HAM, LAS, LISCL,
PAT1, SCR, SCL3, SHR and RGL, play important roles in plant growth and development,
axillary meristem formation, root radial patterning, cell maintenance and proliferation,
defense response and stress tolerance. The genes in each tissue play central roles in pitaya
development. The expression of GRAS genes in the pitaya plant comprises 15 tissues,
including flower buds (four stages—FB1 to FB4), flowers (five stages—F1 to F5), pericarp
(three stages—45 days, 65 days, 85 days) and the pulp of the fruit (three stages—29 days,
35 days, 49 days). Of the 45 HuGRAS genes, most were not expressed (Figure 5). We
choose 12 genes that showed significant differential expression in all tissues: HuGRAS-1,
HuGRAS-6,HuGRAS-7,HuGRAS-12,HuGRAS-18,HuGRAS-21,HuGRAS-25,HuGRAS-29,
HuGRAS-34,HuGRAS-35,HuGRAS-37 and HuGRAS-41.
3.6. HuGRAS Proteins Network Analysis
The HuGRAS proteins and their interaction network revealed that the number of proteins
regulated by each predicted gene is significantly different. Among the 45 HuGRAS genes,
27 genes were involved in 215 possible interactions. Based on network analysis, we divided
the interacting genes into four categories: gray (2–5 interactions), yellow (6–10 interactions),
red (11–15 interactions) and green (16–20 interactions). Based on the maximum interaction,
we identified HuGRAS-1,HuGRAS-18,HuGRAS-6,HuGRAS-36 and HuGRAS-39 as hub genes,
shown with green and red color (Figure 6). In the yellow category, we found that HuGRAS-12,
HuGRAS-29,HuGRAS-35 and HuGRAS-37 interacted significantly with other genes.

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Figure5.TheexpressionheatmapofHuGRASgenesindifferentpitayatissues.Inthisheatmap,15
rowsrepresenttheexpressionsofdifferenttissues,and45columnsrepresentthegenes.Fourflower
budstagesareshownasFB1toFB4,andfiveflowerstagesareshownasF1toF5.Threepericarp
stagesareshownasperiC‐45d,periC‐65dandperiC‐85d,andthreepulpstagesareshownaspul‐
29d,pul‐35dandpul‐49d.Colorchangesfromlightbluetodarkblueshowlessornoexpressionof
HuGRASgenes.Lightyellowtoadarkredcolorshowslessexpressiontoahighlevelofexpression
ofthesegenes.
3.6.HuGRASProteinsNetworkAnalysis
TheHuGRASproteinsandtheirinteractionnetworkrevealedthatthenumberofpro‐
teinsregulatedbyeachpredictedgeneissignificantlydifferent.Amongthe45HuGRAS
genes,27geneswereinvolvedin215possibleinteractions.Basedonnetworkanalysis,we
dividedtheinteractinggenesintofourcategories:gray(2–5interactions),yellow(6–10
interactions),red(11–15interactions)andgreen(16–20interactions).Basedonthemaxi‐
muminteraction,weidentifiedHuGRAS‐1,HuGRAS‐18,HuGRAS‐6,HuGRAS‐36and
HuGRAS‐39ashubgenes,shownwithgreenandredcolor(Figure6).Intheyellowcate‐
gory,wefoundthatHuGRAS‐12,HuGRAS‐29,HuGRAS‐35andHuGRAS‐37interacted
significantlywithothergenes.
Figure 5.
The expression heatmap of HuGRAS genes in different pitaya tissues. In this heatmap,
15 rows represent the expressions of different tissues, and 45 columns represent the genes. Four
flower bud stages are shown as FB1 to FB4, and five flower stages are shown as F1 to F5. Three
pericarp stages are shown as periC-45d, periC-65d and periC-85d, and three pulp stages are shown as
pul-29d, pul-35d and pul-49d. Color changes from light blue to dark blue show less or no expression
of HuGRAS genes. Light yellow to a dark red color shows less expression to a high level of expression
of these genes.
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Figure6.Proteininteractionnetwork.Greenandredgenesaredesignatedashubgenesbecause
theyinteractwithmorethan10genes.Differentcolorsshowtheinteractionsofthegenesasfollows:
green(16–20),red(11–15),yellow(6–10)andgray(2–5).
3.7.IdentificationofCisactingElementsinHuGRASPromoterSequences
Toidentifythebiologicalfunctions(stressresponse,growthanddevelopment)ofthe
HuGRASgenes,all45HuGRASgenesequences(2000bpupstreamofstartcodon)were
selectedforcis‐elementanalysisusingthePlantCAREwebtool(SupplementaryFileS3).
Intotal,17cis‐elementswererecordedinthisstudy(FigureS3).Thecis‐regulatoryele‐
mentsof45GRASproteinsareshowninFigureS3.Ninegenesthatexhibitedhigherex‐
pressionamongtheGRASgenefamilyinthepitayaplantexhibitedvariouscis‐actingreg‐
ulatoryelements,asshowninFigure7.Fourteencis‐actingelementswerecategorizedinto
fourgroups:light‐responsiveelements,growthanddevelopmentelements,stress‐and
defense‐responsiveelementsandhormone‐responsiveelements.
Figure 6.
Protein interaction network. Green and red genes are designated as hub genes because
they interact with more than 10 genes. Different colors show the interactions of the genes as follows:
green (16–20), red (11–15), yellow (6–10) and gray (2–5).

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3.7. Identification of Cisacting Elements in HuGRAS Promoter Sequences
To identify the biological functions (stress response, growth and development) of the
HuGRAS genes, all 45 HuGRAS gene sequences (2000 bp upstream of start codon) were
selected for cis-element analysis using the PlantCARE web tool (Supplementary File S3). In
total, 17 cis-elements were recorded in this study (Figure S3). The cis-regulatory elements of
45 GRAS proteins are shown in Figure S3. Nine genes that exhibited higher expression among
the GRAS gene family in the pitaya plant exhibited various cis-acting regulatory elements,
as shown in Figure 7. Fourteen cis-acting elements were categorized into four groups: light-
responsive elements, growth and development elements, stress- and defense-responsive
elements and hormone-responsive elements.
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Figure7.Thecis‐actingelementsofthepromoterregions(2000bpupstreamofstartcodon)ofnine
HuGRASgenes.
3.8.ExpressionofHuGRASGenesatDevelopmentalStagesofPitaya
Toconfirmtheexpressionsofthepredictedgenesfromthetranscriptomedata,we
conductedqRT‐PCRforHuGRAS‐1,HuGRAS‐6,HuGRAS‐7,HuGRAS‐12,HuGRAS‐18,
HuGRAS‐21,HuGRAS‐25,HuGRAS‐29,HuGRAS‐34,HuGRAS‐35,HuGRAS‐41and
HuGRAS‐37.Wedesignedprimersforthe12candidategenes,categorizedinsixGRAS
subfamilies(SupplementaryFileS4).TheresultsthereofexhibitedthatHuGRAS‐1,
HuGRAS‐7,HuGRAS‐12,HuGRAS‐18,HuGRAS‐25,HuGRAS‐34,HuGRAS‐35,HuGRAS‐
41andHuGRAS‐37showedhigherlevelsofexpressionacrossthetissues(Figure8).The
expressionlevelsoftheHuGRASmembersvariedwidelyindifferenttissues.The
HuGRAS‐1gene,categorizedintheDELLAsubfamily,wassignificantlyexpressedacross
thetissues,includingthestems,FBsandpericarp.However,relativelyweakerexpression
wasobservedinthepulpofthefruit.AmongthePAT1subfamilymembers,HuGRAS‐34,
HuGRAS‐35andHuGARS‐41exhibitedstrongexpressionintheplanttissuesascompared
toHuGRAS‐6,whichexhibitedweakexpressioninthepericarpandthepulp.HuGRAS‐7,
whichbelongstotheSCL‐3subfamily,wasexpressedatalowlevelintheone‐month‐old
stemcellsbutabundantinothertissues.HuGRAS‐12,agenecategorizedintheSCRsub‐
family,wasexpressedatahigherlevelinothertissuesthantheflowerbuds.HuGRAS‐21
andHuGRAS‐29,membersoftheLISCLsubfamily,wereexpressedatlowerlevelsthan
theHuGRAS‐18andHuGRAS‐25groupedinthesamesubfamily,whichwereexpressed
athigherlevelsintheflowerbuds,thepericarpandthepulpofthepitayaplant.HuGRAS‐
37,groupedintotheHAMsubfamily,washighlyexpressedintheflowerbudsbutweakly
expressedintheone‐month‐oldstems.Ninegenes,whichwerecategorizedintosixsub‐
families,exhibitedhigherexpressionlevelsandmightplaykeyrolesinthegrowthand
developmentofthepitayaplant.
Figure 7.
The cis-acting elements of the promoter regions (2000 bp upstream of start codon) of nine
HuGRAS genes.
3.8. Expression of HuGRAS Genes at Developmental Stages of Pitaya
To confirm the expressions of the predicted genes from the transcriptome data, we
conducted qRT-PCR for HuGRAS-1,HuGRAS-6,HuGRAS-7,HuGRAS-12,
HuGRAS-18
,
HuGRAS-21,HuGRAS-25,HuGRAS-29,HuGRAS-34,HuGRAS-35,HuGRAS-41 and HuGRAS-
37. We designed primers for the 12 candidate genes, categorized in six GRAS subfami-
lies (Supplementary File S4). The results thereof exhibited that HuGRAS-1,HuGRAS-
7,HuGRAS-12,HuGRAS-18,HuGRAS-25,HuGRAS-34,HuGRAS-35,HuGRAS-41 and
HuGRAS-37 showed higher levels of expression across the tissues (Figure 8). The ex-
pression levels of the HuGRAS members varied widely in different tissues. The HuGRAS-1
gene, categorized in the DELLA subfamily, was significantly expressed across the tissues, in-
cluding the stems, FBs and pericarp. However, relatively weaker expression was observed
in the pulp of the fruit. Among the PAT1 subfamily members, HuGRAS-34,HuGRAS-35
and HuGARS-41 exhibited strong expression in the plant tissues as compared to HuGRAS-6,
which exhibited weak expression in the pericarp and the pulp. HuGRAS-7, which belongs
to the SCL-3 subfamily, was expressed at a low level in the one-month-old stem cells but
abundant in other tissues. HuGRAS-12, a gene categorized in the SCR subfamily, was ex-
pressed at a higher level in other tissues than the flower buds. HuGRAS-21 and HuGRAS-29,
members of the LISCL subfamily, were expressed at lower levels than the HuGRAS-18 and
HuGRAS-25 grouped in the same subfamily, which were expressed at higher levels in the
flower buds, the pericarp and the pulp of the pitaya plant. HuGRAS-37, grouped into the
HAM subfamily, was highly expressed in the flower buds but weakly expressed in the
one-month-old stems. Nine genes, which were categorized into six subfamilies, exhibited

Biology 2023,12, 11 12 of 17
higher expression levels and might play key roles in the growth and development of the
pitaya plant.
Biology2023,12,xFORPEERREVIEW14of20



Figure8.qRT‐PCRexpressionanalysisof12genesinsixtissuesofthepitaya(S.undatusL.)plant.
TheX‐axisrepresentstheplanttissues,includingone‐month‐oldstem(S1M),one‐year‐oldstem
(S1Y),two‐year‐oldstem(S2Y),flowerbud(FB),pericarp(PeriC)andpulp.Errorbarsrepresentthe
standarddeviationsforthethreereplicates.
4.Discussion
Pitaya(S.undatusL.)isatropicalfruit,typicallycacti,evergreen,andconsistsofclad‐
odes(amodifiedstemreplacestheleavesforphotosynthesisfunction)whichperformits
functioningasaleaf.Theflowersandfruitsareedible,andthepericarpandpulpofS.
undatusarewhiteincolor.Thefruitsarehighlyenrichedwithpolyphenols,tannis,beta‐
lainsandnonbetalainicandantioxidantcompounds[66].Duetotheimportanceofthe
pitayatropicalfruit,thepresentstudywascarriedouttoexplorethegrowthandthede‐
velopmentalprocessoftheplant.TheGRASTFisbeingexploredinothercropspecies,
suchasArabidopsis[55],Medicagotruncatula[48],pepper[54],cotton[50],soybeans[27],
tomatoes[67],Chinesecabbage[16]andtropicalfruitsuchaslitchi[28],butwecouldnot
findanyresearchstudiesabouttheGRASgenefamilyinS.undatusL.GRASproteinshave
beenrecognizedasimportantTF,playingdifferentfunctionsinplantgrowthanddevel‐
opment,includingpatterningofrootsandshoots,responsestovariouskindsofstresses,
stem‐cellinitiationandmaintenance[35],lightsignalingandthegibberellic‐acidsignal‐
transductionpathway[21,68].
Withtheavailabilityofthepitayareferencegenome[2]andpitayatissueexpression
dataviathepitayagenomeandmultiomicsdatabase[56],weperformedagenome‐wide
identificationoftheGRASgenefamilymembersinthepitayagenome.Inthecurrent
study,wefound45GRASgenefamilymembersinthisgenome,namedHuGRAS‐1to
HuGRAS‐45;theywerewidelydistributedon11chromosomes(Figure4).Mostofthe
HuGRASgeneswerefoundwereontheendsofthesechromosomes,whichisinaccord‐
ancewithotherplantspecies,suchaswatermelon,potatoes,riceandArabidopsis[69].
Theconservedmotifstructures(Figure3)andHuGRASgenesequences(FigureS2)exhib‐
itedthesamepatternofconservedmotifsandexon–intronsequences,respectively,
Figure 8.
qRT-PCR expression analysis of 12 genes in six tissues of the pitaya (S. undatus L.) plant.
The X-axis represents the plant tissues, including one-month-old stem (S1M), one-year-old stem
(S1Y), two-year-old stem (S2Y), flower bud (FB), pericarp (PeriC) and pulp. Error bars represent the
standard deviations for the three replicates.
4. Discussion
Pitaya (S. undatus L.) is a tropical fruit, typically cacti, evergreen, and consists of
cladodes (a modified stem replaces the leaves for photosynthesis function) which perform
its functioning as a leaf. The flowers and fruits are edible, and the pericarp and pulp
of S. undatus are white in color. The fruits are highly enriched with polyphenols, tannis,
betalains and nonbetalainic and antioxidant compounds [
66
]. Due to the importance of
the pitaya tropical fruit, the present study was carried out to explore the growth and the
developmental process of the plant. The GRAS TF is being explored in other crop species,
such as Arabidopsis [
55
], Medicago truncatula [
48
], pepper [
54
], cotton [
50
], soybeans [
27
],
tomatoes [
67
], Chinese cabbage [
16
] and tropical fruit such as litchi [
28
], but we could
not find any research studies about the GRAS gene family in S. undatus L. GRAS proteins
have been recognized as important TF, playing different functions in plant growth and
development, including patterning of roots and shoots, responses to various kinds of
stresses, stem-cell initiation and maintenance [
35
], light signaling and the gibberellic-acid
signal-transduction pathway [21,68].
With the availability of the pitaya reference genome [
2
] and pitaya tissue expression
data via the pitaya genome and multiomics database [
56
], we performed a genome-wide
identification of the GRAS gene family members in the pitaya genome. In the current study,
we found 45 GRAS gene family members in this genome, named HuGRAS-1 to HuGRAS-45;
they were widely distributed on 11 chromosomes (Figure 4). Most of the HuGRAS genes
were found were on the ends of these chromosomes, which is in accordance with other
plant species, such as watermelon, potatoes, rice and Arabidopsis [
69
]. The conserved
motif structures (Figure 3) and HuGRAS gene sequences (Figure S2) exhibited the same
pattern of conserved motifs and exon–intron sequences, respectively, suggesting that these
genes may have similar functions to those reported in previous studies [21].

Biology 2023,12, 11 13 of 17
In accordance with phylogenetic analysis, we compared the 45 HuGRAS gene se-
quences with 335 sequences of GRAS proteins from maize, soybeans, Medicago truncatula,
rice, Arabidopsis and tomatoes. HuGRAS genes were divided into nine subfamilies based
on clade support values: PAT1, SHR, LISCL, HAM, SCR, RGL, LAS, DELLA and SCL3
(Figure 2). Each subfamily carried varying numbers of HuGRAS genes, and the PAT1
subfamily contained the largest number of HuGRAS genes. The protein sequences and
differential expression profiles of pitaya tissues aid in the identification of the genes that
play key roles in growth and development. Expression and network analysis provide a
clue to locating genes that exhibit high levels of expression (Figure 5) and interact with
many other genes (Figure 6). With the help of expression analysis and network analysis,
12 selected genes were categorized into their respective subfamilies, as predicted in the
phylogenetic tree (Figure 2). All genes were placed in their respective GRAS families:
HuGRAS-1 in the DELLA subfamily; HuGRAS-6,HuGRAS-34,HuGRAS-35 and HuGRAS-41
in the PAT1 subfamily; HuGRAS-7 in the SCL-3 subfamily; HuGRAS-12 in the SCR sub-
family; HuGRAS-18,HuGRAS-21,HuGRAS-25 and HuGRAS-29 in the LISCL subfamily;
and HuGRAS-37 in the HAM subfamily. qRT-PCR was carried out for the predicted gene
subfamilies (Figure 8) to confirm their expressions in different stages of the plant. The
HuGRAS-1 gene, categorized in the DELLA subfamily, was significantly expressed across
the tissues, as the DELLA subfamily is involved in the growth and development of the
plant [
41
]. In the absence of gibberellic acid, DELLA proteins interact with light-responsive
TFs, including phytochrome-interacting factors (PIFs), to form inactive complexes [
70
],
while higher expression of gibberellic acid degrades DELLA proteins and initiates the
growth rate [
71
]. Our network analysis revealed that the HuGRAS-1 gene interacts with
almost 20 other proteins, so our results are consistent with the prediction of Hirsch and
Oldroyd, 2009 [
41
] that DELLA proteins interact with other PIF families and make com-
plexes with them. This DELLA–PIF TF complex is possibly competitive but a common
mechanism for DELLAs to make complexes for light- and gibberellic-acid-signaling to
alter environmental conditions [
41
]. DELLA proteins also regulate immune responses
by regulating the jasmonic- and salicylic-acid pathways. The PAT1 subfamily members,
HuGRAS-34,HuGRAS-35 and HuGARS-41, exhibited strong expressions in plant tissues as
compared to HuGRAS-6, which was weakly expressed. PAT1 is a specific member of the
GRAS family that interacts with light signaling via phytochrome A to regulate the plant
developmental process, including de-etiolation and hypocotyl elongation [
45
]. HuGRAS-7,
which belongs to the SCL-3 subfamily, was expressed at a low level in one-month-old
stem cells but exhibited higher expression abundantly in other tissues. The SCL-3 subfam-
ily acts antagonistically, downstream to gibberellic-acid DELLA responses and upstream
to gibberellic-acid-biosynthesis pathways, during plant growth and development [
47
].
HuGRAS-12 was categorized in the SCL-3 subfamily and was expressed at a higher level in
other tissues than flower buds. HuGRAS-37 is grouped into the HAM subfamily and was
highly expressed in flower buds but weakly expressed in one-month-old stems. The SCR
and HAM subfamilies play key roles in root/shoot patterning and cell differentiation in
shoot meristem maintenance [
72
]. HuGRAS-21 and HuGRAS-29, members of the LISCL sub-
family, were expressed at lower levels than the HuGRAS-18 and HuGRAS-25 genes of the
same subfamily, which were expressed at a higher level in the flower buds, the pericarp and
the pulp of the pitaya plant. Higher levels of expression of LISCL genes (HuGRAS-18 and
HuGRAS-25) may predict their role in flower development and fruit ripening. The LISCL
subfamily of the GRAS protein has been reported to play a role in another development,
of Lilium longiflorum L. [
73
]. Previous studies have revealed that redundancy of relative
expression and phytohormones in different parts of the reproductive tissue (panicle) can
lead to defects in growth. Similarly, varying expression levels of GRAS-family genes might
also have different functions in different pitaya tissues [74,75].
In this study, we analyzed the GRAS TF family in the pitaya plant (S. undatus L.)
and six other species, including maize, soybeans, Medicago truncatula, rice, Arabidopsis
and tomatoes. A total of 380 GRAS genes were analyzed in this research, in addition to

Biology 2023,12, 11 14 of 17
45 genes that were predicted from the pitaya genome. We categorized these genes into
nine subfamilies based on phylogenetics and previous studies of other crops. Among
the nine subfamilies of GRAS, few genes showed higher expression in different tissues
of pitaya plant. These genes were categorized into six sub-families including DELLA
(HuGRAS-1), SCL-3 (HuGRAS-7), PAT1 (HuGRAS-34,HuGRAS-35,HuGRAS-41), HAM
(HuGRAS-37), SCR (HuGRAS-12) and LISCL (HuGRAS-18,HuGRAS-25) which may have
potential key role in the growth and development of the pitaya plant. Their roles were
also confirmed using in silico cis-acting analysis (Figure S3). As we could see, cis-acting
elements, including gibberellin, auxin, ABA, jasmonic-acid and salicylic-acid-responsive
elements were abundantly present in the HuGRAS promoters. These genes can be used
to study the regulatory pathways of specific plant traits. Positive and negative regulators
can be identified from the pathways; then the CRISPR system can be used to produce a
transgene-free pitaya plant. Previously, many crops have been improved using the latest
genome editing technique [
76
,
77
]. Collectively, our results lay a theoretical foundation for
the role of GRAS genes in pitaya growth and development. It provides valuable information
to improve the pitaya breeding program.
5. Conclusions
This study is the first comprehensive genome-wide identification of the GRAS gene
family in pitaya (S. undatus L.). This research might aid in the interpretation of the GRAS
genes function, protein interactions, signaling-pathway regulations and expression pat-
terns in different tissues. The comparative study between the GRAS families of six species,
the phylogenetic tree, the expression pattern and the gene network analysis will lay a
foundation for the functional characterization of the genes in pitaya. Understanding the
possible roles of nine predicted genes (HuGRAS-1,HuGRAS-7,HuGRAS-12,HuGRAS-18,
HuGRAS-25
,HuGRAS-34,HuGRAS-35,HuGRAS-37,HuGRAS-41) from the six subfamilies
of GRAS gene and their expression patterns in different tissues provides insightful infor-
mation for the development of pitaya fruit’s economic, agronomic and ecological benefits.
Altogether, the current study is the first report on the GRAS gene family in pitaya tropical
fruit. The identification of the genes will assist in clarifying the molecular genetic basis and
aid in improving the genotypes in the breeding program.
Supplementary Materials:
The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/biology12010011/s1.
Author Contributions:
Conceptualization, Q.U.Z.; methodology, Q.U.Z.; formal analysis, Q.U.Z.;
investigation, Q.U.Z., M.A.H., L.U.K. and H.-F.W.; data curation, Q.U.Z.; writing—original draft
preparation, Q.U.Z.; writing—review and editing, Q.U.Z., M.A.H., L.U.K., J.-P.C., D.K., L.H. and
W.L. supervision, H.-F.W.; project administration, H.-F.W. All authors have read and agreed to the
published version of the manuscript.
Funding:
This study was funded by the Hainan Province Science and Technology Special Fund
(ZDYF2022XDNY190), the Project of Sanya Yazhou Bay Science and Technology City (Grant Number:
SCKJ-JYRC-2022-83) and the Hainan Provincial Natural Science Foundation of China (421RC486).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data generated and analyzed in this study are available in the
Supplementary Materials.
Acknowledgments: The authors thank all of the subjects who participated in this study.
Conflicts of Interest: The authors declare no conflict of interest.
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