Introduction

Sunflower (Helianthus annuus) belongs to compositae family and is native to North America (Schilling and Heiser 1981; Blackman et al. 2011). This crop includes diploid, tetraploid and hexaploid species having basic set of chromosomes 17 (Rieseberg and Seiler 1990). It is an important oil seed crop and complementary source of protein for human being, dairy and livestock animals. Sunflower is also used for ornamental purpose and is a source of chemical feed stock. Sunflower genome was sequenced in 2017 and reportedly has estimated genome size of 36 gigabases (Badouin et al. 2017).

Cuticular waxes are mixture of long chain fatty acids and their derivatives (Shaheenuzzamn et al. 2019; Alfarhan et al. 2020). Basic components of plant cuticular waxes are aldehydes, alkanes, fatty acids, ketones, acetones, wax esters, terpenoids and sterols (Shaheenuzzamn et al. 2019). Biochemical mechanism of wax elongation is fully characterized, however very less information is available about the proteins involved in wax biosynthesis process after 26 carbons (Pascal et al. 2013). Cuticular wax seals the areal parts of land plants to prevent them from non-stomatal water loss (Ahmad et al. 2015; Alfarhan et al. 2020). Leaf glaucousness is a trait referred to as plants adaptation to drought (Islam et al. 2009).  Reduction in wax quantity exhibits high transpiration rate where as high water loss has been observed in low waxy leaves (Alfarhan et al. 2020). Cuticular waxes resist plants against insects, pathogens and bacteria (Zeisler-Diehl et al. 2018), protect plants from ultraviolet radiations (Laila et al. 2017; Alfarhan et al. 2020), decrease water deposition on plant surface, reduce the retention of dust, air pollutants and pollens (Wang et al. 2019). Wax biosynthesis process starts with elongation of 16:0 Acyl-CoA to very long chain fatty acid which is reduced to primary alcohol and formation of alkyl ester (Lai et al. 2007).

Long-chain acyl-CoA synthetase (LACS) has a critical role in biosynthesis of all fatty acid derived molecules particularly in cuticular wax and cutin biosynthesis pathways (Lü et al. 2009). LACS esterifies free fatty acids to acyl-CoAs, a key activation step that is necessary for the utilization of fatty acids by most lipid metabolic enzymes (Lü et al. 2009; Pulsifer et al. 2012). Sometime LACS enzyme expresses dual role in activation of long chain fatty acids for synthesis of cellular lipids and their degradation via beta-oxidation (Jenks et al. 1995). Biosynthesis mechanism of cuticular waxes begins with the synthesis of C16 and C18 long chain fatty acids (LCFA) in plastids (Ahmad et al. 2015; Shaheenuzzamn et al. 2019). These LCFAs are then transported to cytoplasm where coenzyme (CoA) activate them by long chain acyl-CoA synthetases (Schnurr et al. 2004; Samuels et al. 2008).

Cuticular wax is a lipid-based barrier to seal the areal surface of land plants and play protective role (Alfarhan et al. 2020). LACS1 gene functions as very long chain acyle-CoA synthetase during wax metabolism (Lü et al. 2009). Similarly, LACS2 gene have overlapping function with LACS1 in cutin and wax synthesis (Pulsifer et al. 2012). In A. thaliana, LACS1, LACS2 and LACS3 are expected to be cuticle biosynthesis genes (Pulsifer et al. 2012). They are also expressed for intracellular trafficking and transmembrane transport (Pulsifer et al. 2012). LACS6 gene acts in both the wax and cutin biosynthesis pathways preferentially uses palmitoleate, palmitate, linoleate and eicosenoate (Lü et al. 2009) and seems to have a specific activity against very long-chain fatty acid (VLCFA) class with acids longer than 24 carbons (Lü et al. 2009). LACS6 also show redundant function when it expressed with LACS7 during seed development process (Shockey et al. 2002).

The aim of present study was genome-wide analysis of LACS gene family in sunflower. Further genomic comparison was performed between Arabidopsis and sunflower to find the functional similarities in them by using different bioinformatics tools. To explore the role of LACS family for wax biosynthesis genes in sunflower, we subjected the sunflower genotypes to drought stress and their expression profile was studied.

Materials and Methods

Retrieval of protein sequences

Protein sequences of A. thaliana LACS genes were retrieved from “The Arabidopsis Information Resource” (TAIR) (https://www.arabidopsis.org/). As, no LACS gene has been characterized in H. annuus, hence pblast program at NCBI (https://www.ncbi.nlm.nih.gov/) was used to obtain the similar sequences in this crop. These sequences were further verified at Plant Genome and System Biology (PGSB) databases (https://pgsb.helmholtz-muenchen.de/plant/plantsdb.jsp) and Phytozome v. 11.0 (https://phytozome.jgi.doe.gov/pz/portal.html) database.

Physio-chemical properties of LACS proteins and subcellular locations

Different physio chemical properties such as exon numbers, amino acid length, molecular weight, isoelectric point of LACS proteins in A. thaliana and H. annuus were computed by using online web tool “protpram” on Expasy (https://web.expasy.org/cgi-bin/protparam/protparam) according to Gasteiger et al. (2005). Sub cellular location of LACS genes in Arabidopsis and sunflower was determined according to Chou and Shen (2010) using server (Plant-mPLoc https://www.csbio.sjtu.edu.cn/bioinf/plant/).

Sequence alignment and construction of phylogenetic tree

LACS protein sequences of A. thaliana, H. annuus and Z. mays were aligned by online tool ClustalX (Sun et al. 2015). Aligned sequences were used for construction of phylogenetic tree according to neighbor joining method (Saitou and Nei 1987) by using MEGA 5.2 program (Tamura et al. 2011) at 1000 boost strap value.

Conserved motifs, gene structure analysis and chromosomal mapping

To further study the structure of LACS proteins, conserved motif analysis of LACS proteins was carried out by using MEME SUIT 4.9.1 tool (https://meme.nbcr.net/meme/cgi-bin/meme.cgi) with their default parameters. Intron and exon organization in both plant species was discovered by using Gene Structure Display Server (GSDS) 2.0 (https://gsds.cbi.pku.edu.cn/). Chromosomal mapping of A. thaliana genes were performed by using chromosome map tool at The Arabidopsis Information Resource (TAIR) (https://www.arabidopsis.org/jsp/ChromosomeMap/tool.jsp) whereas in H. annuus exact location of LCAS genes were mapped by using excel sheet.

Prediction of evolutionary history and protein-protein interaction

To predict the evolutionary history of these genes, protein sequences of A. thaliana and H. annuus were submitted to online synteny tool Circoletto (tools.bat.infspire.org/circoletto). The predicted protein–protein interaction (PPI) map of LACS proteins was generated from the STRING database (https://string-db.org/cgi/my.pl?sessionId=1Ye2FGXxwMVL) (Szklarczyk et al. 2011).

Plants material and drought treatment

To analyze the expression pattern of HanLACS1 and HanLACS3 genes in sunflower under drought conditions, four sunflower genotypes Hysun-33, FH-331, FH-629 and FH-630 obtained from Oilseed Research Institute, Ayyub Agriculture Research Institute, Faisalabad, were cultivated in pots in growth chambers, containing red sandy soil and manure (2:1) with a program set to 25/22°C (day/night), 16-h photoperiod, and relative humidity of 75%. At the age of 30 days’ plants were subjected to drought stress by withholding water for ten days. Samples from both treated and non-treated plants were collected for three biological replicates and were frozen immediately in liquid nitrogen at -80°C until further analysis.

RNA isolation and RT-qPCR analysis

Total RNA from frozen samples was isolated by using TriZol reagents according to the manufacturer’s instructions. RNA concentration was measured with nanodrop, ND-1000 (Nano Drop Technologies, Inc.), spectrophotometer using the nucleic acid program. Primers were designed from a list of genes belonging to LACS gene family involved in epicuticular wax biosynthesis based on previous studies by using online tool primer3 (https://frodo.wi.mit.edu/). Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used for first strand cDNA synthesis. Gene expression levels were studied by quantitative real time PCR using SYBER Green qPCR Master Mix (ThermoFisher Scientific, U.S.A.) in CFX96 Real-Time PCR System (BIO-RAD, U.S.A.). The variations in gene expression were calculated using the 2^−ΔΔCt analysis method. The quantification was carried out by the Actin gene as a reference gene. The specific primers for LACS genes used in qPCR could be seen in Table 1.

Results

Identification of LACS genes, physio-chemical properties and subcellular locations

Previously LACS family has not been characterized in sunflower due to un-availability of genome sequence. Information about the physiochemical characteristics of A. thaliana are shown in Table 2. Chromosomal number indicated that LACS genes were present on all the five Arabidopsis chromosomes and exon number varied from 12 to 23. It was observed that length of genomic DNA was ranged from 3480 bp (LACS8-2) to 5609 bp (LACS6-1). The number of amino acids ranged from 522 (LACS2-2) to 720 (LACS8-1, LACS8-2). Predicted molecular weight varied from 78342.6 to 57634.5. The theoretical isoelectric point was ranging from 5.63 to 8.01 indicating that some proteins are basics, and some were acidic in nature. Subcellular location showed that out of sixteen proteins six were present in endoplasmic reticulum, six in chloroplast, one each in plasma membrane, nucleus, golgi apparatus and mitochondria.

In H. annuus, exon numbers were counted from 5 (LACS3-1) to 23 (LACS6-1). Amino acid length of LACS proteins in this crop species varied from 95 (LOC110868952) to 720 (HannXRQ_Chr04g0126391). Genomic length was in range of 1117bp to 18099 bp (Table 3). Molecular weight of proteins was diversified from 10985.88 kDa to 78573.77kDa. Isoelectric point of sunflower LACS proteins (pI) was in between from 5.37 to 8.49. subcellular location indicated that seven proteins were present in chloroplast, five in plasma membrane, four in golgi apparatus, and two in nucleus and one in peroxisomes.

Phylogenetic relationship of LACS proteins in A. thaliana and H. annuus

To study the phylogenetic relationship of LACS proteins in A. thaliana, H. annuus and Z. mays a phylogenetic tree was constructed by multiple sequence alignment of these proteins (Fig. 1). Phylogenetic tree divided LACS proteins in seven subgroups. These clusters contained 13, 8, 12, 8, 7, 4 and 5 members respectively. First cluster was the largest than others with thirteen members and sixth was the smallest. It was observed that first five clads contained protein members from all three species suggesting that these proteins are evolutionary conserved. Our results are in line with (Azeem et al. 2018; Waqas et al. 2019) who reported similar results in chickpea. 6^th clad possessed only Arabidopsis proteins and 7^th clad was belonging to H. annuus proteins only which mean that these proteins may be evolutionary diverse from each other.

Gene structure analysis of LACS genes in A. thaliana and H. annuus

To get the information regarding intron and exon organization, their number and length in LACS gene families of A. thaliana and H. annuus, genomic DNA and coding sequences were analysed by using GSDS 2.0 server. The results revealed that intron-exon structures are conserved within groups of LACS genes (Fig. 2). Maximum number of exons was present in HaLACS6-1 which was 23. According to intron-exon length HaLACS6-3 was the smallest gene which contained only three exons. Further it was observed that some genes clustered together having similar numbers and length of CDSs, even they showed variation in length of introns and untranslated regions (UTRs). Two genes, HaLACS8-2 and HaLACS9-2 were closely related to each other as they fall in same cluster and have similar nature of intron-exon organization throughout the genome. Similarly, AtLACS8-1, AtLACS7-1 and AtLACS6-1 have homology among them and fall in same group. Similarity in intron/exon organization within a subgroup of phylogenetic tree has been reported by (Bari et al. 2018; Waqas et al. 2019).

Conserved motif analysis for LACS proteins in A. thaliana and H. annuus

The results of conserved motifs were presented in Fig. 3. Motif analysis showed ten different conserved motifs in Arabidopsis and sunflower in LACS proteins. Maximum 11 motifs were recorded on Arabidopsis protein LACS 6-1 while minimum 1 motif was noticed on sunflower protein LACS6-2. It was also noted that pattern of conserved motifs was almost same with in a clad of phylogenetic tree. As previously no conserved motif analysis was available in sunflower hence, we were unable to compare our results.

Chromosomal mapping of LACS genes in A. thaliana and H. annuus

Table 1: List of primers used for qRT-PCR

Table 2: Different Physio-chemical characteristics of LACS genes and their homologues showing variability in A. thaliana

Where E.R= Endoplasmic reticulum, Ch.P= Chloroplast, P.Mem= Plasma membrane, Nuc= Nucleus, Mito.C= Mitochondria and Gol.A= Golgi apparatus

Table 3: Different Physio-chemical characteristics of LACS genes and their homologues showing variability in H. annuus

Where, Ch.P= Chloroplast, P.Mem= Plasma membrane, Nuc= Nucleus, Per.Oxi= Peroxisome and Gol.A= Golgi apparatus

The results of Fig. 4 (a) indicated that in Arabidopsis, LACS genes were present on all the five chromosomes. Maximum three genes were present on different locations at 1^st chromosome. In sunflower genome LACS genes were unevenly distributed on seventeen chromosomes. It was noted that maximum four genes were located on 7^th chromosome however no LACS gene was present on chromosome 2, 5, 12, 14, 16 and 17. Distribution of LACS genes in both plant species is showen in Fig. 4 (a, b).

Evolutionary relationship of Arabidopsis and Sunflower LACS genes

Evolutionary relationship among different species showed that weather these species were from same origin or not. It also enables us to know about their origin and ecosystem. High similarity indicates that these species were from same origin and similar environmental conditions whereas low similarity represents the contrasting environmental conditions. A comparative synteny analysis was performed to get the idea about the evolutionary relationship and `origin of LACS gene family in Arabidopsis and Sunflower. Synteny analysis was performed by using 16 Arabidopsis and 19 sunflower LACS proteins. The results showed that both these species were closely related to each other in their origin as per Fig. 5. Arabidopsis LACS gene AtLACS9-1 syntenic to sunflower HaLACS9-1, HaLACS9-2, HaLACS8-1 and HaLACS8-2. Similarly, Arabidopsis LACS1, LACS2 were syntenic to sunflower LACS1 and LACS2 respectively. As previously no study was available in this regard hence results remained un-compared.

Fig. 1: Phylogenetic tree of LACS proteins. The tree was constructed with amino acid sequences of A. thaliana, H. annuus and Z. mays using neighbor joining method at boost strap value of 1000 replicates. Sequences were aligned with ClustalX and tree was constructed using MEGA 5.2 program

Fig. 2: Phylogenetic relationship and intron-exon organization of A. thaliana and H. annuus LACS genes. Yellow and blue boxes representing CDS and UTRs respectively and introns are represented by black lines. The analysis was performed by GSDS 2.0

Fig. 3: Motif analysis of A. thaliana and H. annuus LACS genes. The colored box in each line represent motif. The non-conserved motifs are represented by blank lines

Fig. 4: Chromosomal mapping of LACS gene family in A. thaliana and H. annuus. Asterisks indicate the positions of genes on each chromosome

Fig. 5: Synteny analysis of LACS genes between Arabidopsis and sunflower. Colored lines which connect two regions indicate syntenic regions between Arabidopsis and sunflower

Fig. 6: Protein-protein interaction analysis of LACS proteins. The analysis was carried out using online server STRING

Fig. 7: Effects of drought stress on the expression of LACS1 and LACS3 in sunflower. The expression levels were examined by qRT-PCR. The results are means of three biological replications. Untreated plants were used as control

Prediction of LACS protein–protein interaction network

Protein-protein interaction analysis was carried out to reveal the unforeseen and unique functional role of well characterized protein. This interaction also explored that at which conditions these proteins interact and what are the functional implications of these interactions. So, to clarify the interaction among LACS1, LACS2, LACS3, LACS4, LACS5, LACS6, LACS7, LACS8 and LACS9 proteins in Arabidopsis, protein–protein interaction network was predicted using STRING online tool. The results (Fig. 6) showed nine number of nodes, 36 number of edges, average node degree was 8, average local clustering coefficient was one and PPI enrichment P-value:< 1.0e-16.

Expression analysis of LACS1 and LACS3 genes in sunflower

We performed the Quantitative RT-PCR analysis to detect the expression of LACS genes in four sunflower cultivars by subjecting these cultivars under drought stress. Then expression of these genes was compared with normally watered plants. Expression analysis of LACS genes indicated that these genes showed their expression in all the four cultivars. LACS1 showed its expression in three (FH-629, FH-630 and Hysun-33) cultivars, whereas LACS3 expressed in (FH-331- FH-629 and FH-630) as shown in Fig. 7. It was noticed that drought stress upregulated the expression of under study LACS genes as compared to control. Higher expression of wax biosynthesis genes under drought stress has been observed in Arabidopsis (Seo et al. 2011), rice (Zhou et al. 2013) and wheat (Bi et al. 2017; Zhao et al. 2018).

Discussion

Biotic and abiotic stresses badly effect the growth and development of crop plants causing huge losses to grain yield (Zhou et al. 2020). Cuticular waxes play important role to protect the plants from various biotic and abiotic stresses such as, drought, salinity, cold, ultraviolet radiation, insects, bacteria and pathogens (Ahmad et al. 2015; Shaheenuzzamn et al. 2019). Wax biosynthesis gene families i.e., LACS, CER, KCS, KCR and FAR play important role in biotic and abiotic stress tolerance (Ahmad et al. 2015; Shaheenuzzamn et al. 2019). The role of these gene families has been characterized in Arabidopsis, rice, wheat and maize (Schnurr et al. 2004; Zhu et al. 2014; Shaheenuzzamn et al. 2019). However, no study was regarding sunflower. So, we selected the long chain acyl-CoA synthetase (LACS) gene family to validate its expression in sunflower under drought conditions. LACS converts free fatty acids to acyl-CoA thioesters that play an important role in fatty acid metabolism (Shockey et al. 2002). The other major function of LACS enzyme is fatty acid transportation. LACS enzymes are also involved in various fatty acids derived metabolic pathways i.e., fatty acid β-oxidation, triacylglycerol, phospholipids and jasmonate biosynthesis (Shockey et al. 2002).

In present research, we provided the complete overview of LACS gene family in sunflower. We conducted in silico identifications and explored the potential role of LACS genes in sunflower through computational tools. Further we analyzed the phylogenetic relationship, subcellular location, gene structure, chromosomal location, conserved motifs, and protein-protein interactions, along with expression analysis of LACS gene family under drought conditions. In previous research nine genes of this family AtLACS1 to AtLACS9 have been reported and characterized in Arabidopsis (Shockey et al. 2002). By BLAST analysis of A. thaliana LACS genes, we identified 19 LACS genes along with their small variants in sunflower. Subcellular localization analysis revealed that these genes were present in various cell organelles i.e., endoplasmic reticulum, nucleus, chloroplast, peroxisomes and mitochondria (Table 3).

Presence of LACS proteins in various key organelles of plant cell is the indication of their active participation in cellular metabolism during a biotic stress conditions (Carther et al. 2019). Previously presence of LACS genes in various subcellular locations has been reported by (Browse and Somerville 1991; Shockey et al. 2002; Fulda et al. 2004). A phylogenetic analysis is helpful to understand the evolutionary pattern of many morphological and chemical traits (Soltis and Soltis 2000, 2003). Our phylogenetic analysis gave rise to seven distinct clads (Fig. 1). First cluster was the most complex one and it was observed that HanLACSs were similar to other plant genes in this cluster. It was noted that Arabidopsis, sunflower and maize LACS6, LACS7 genes were falling in same clad and LACS3, LACS4 and LACS5 in same subgroup which supported the results of (Fulda et al. 2004), who reported similar results in Arabidopsis.

The exon-intron distribution can be considered as an imprint of evolution in a gene family, which can provide extra evidence to reveal the phylogenetic relationship of the gene family from different organisms (Yang et al. 2019). In this study LACS genes from Arabidopsis and sunflower showed 3–18 introns (Fig. 2), indicating high structural diversity regarding LACS genes in these plants. During the comparison intron/exon organization of LACS gene between Arabidopsis and sunflower plants, it was observed that genes falling in same cluster probably have same exon numbers. Similarity between genes of different species showed that they came from similar ancestor and these genes were strongly affected by repetitive DNA duplication phenomena during the evolution process (Carther et al. 2019; Lynch and Conery 2000). Chromosomal mapping indicate that these genes were located on chromosomes 1, 2, 3, 6, 7, 8, 9, 10, 12 and 14 (Fig. 4).

Among abiotic stresses, drought is a major limiting factor that effect the plant growth, development and ultimately reduction in plant production (Awan et al. 2015; Javed et al. 2016). Many studies have been conducted to improve the plants adoptability to drought stress viewing root architecture, leaf organization, drought tolerance and avoidance mechanisms, however no prior concentration has been given for the identification of cuticular wax biosynthesis genes and their role under limited water conditions. Expression profile of two LACS genes LACS1 and LACS3 was determined in four sunflower cultivars by using qRT-PCR and their transcript level was determined under drought and normal conditions. LACS1 showed its expression in three cultivars i.e., FH-29, FH-30 and Hysun-33 and LACS3 expressed in FH-331, FH-629 and FH-630. It was observed that expression level of these genes was higher in drought stressed plants as compared to controls which were upregulated under drought conditions and have role in drought tolerance.

Previously, it has been reported that Arabidopsis and yeast LACS1 genes are involved in wax biosynthesis, metabolic pathway, fatty acid and glycerol-lipid metabolism (Shockey et al. 2002; Lü et al. 2009; Pulsifer et al. 2012). LACS1 gene function as very long chain acyl-CoA synthetase during wax metabolism (Kunst and Samuels 2003). Similarly, Arabidopsis LACS3 gene have overlapping function with LACS1 during cutin and wax synthesis (Lü et al. 2009). Variation in LACS gene expression under drought conditions showed their metabolic activities in plant tissues. Similar type of trend for several metabolites was noted in tomato (Zgallai et al. 2005), maize (Guo et al. 2017) and soybean (Carther et al. 2019).

Conclusion

Comprehensive genomic analysis and expression profiling of LACS genes in H. annuus revealed the presence of 19 genes in this species. These genes were located in different chromosomes and were present in various subcellular locations. Phylogenetic and conserved motif analysis confirmed the evolutionary association among sunflower and Arabidopsis LACS genes. Results of qRT-PCR showed that drought stress upregulated the expression of under study LACS genes as compared to control. This information would be helpful for selection of wax and stress responsive candidate genes in sunflower for further studies. Moreover, this is among the pioneer investigations on comparative genomics of wax biosynthesis genes in sunflower that may lead the foundation for further research in this aspect.

Acknowledgements

The authors are thankful to Mr. Fida Hussain, Research Officer, Oilseed Research Institute, Ayyub Agriculture Research Institute, Faisalabad, Pakistan, for the provision of sunflower genotypes to conduct the research.

References

Ahmad HM, Mahmood-ur-Rahman, Q Ali, SI Awan (2015). Plant cuticular waxes: A review on functions, composition, biosyntheses mechanism and transportation. Life Sci J 12:60‒67

Alfarhan AH, R Rajakrishnan, A Mohamed, Al-Shehri, ASM Al-Tamimi, S Al-Obaid, S Khalaf (2020). Analysis of the cuticular wax composition and ecophysiological studies in an arid plant Z. nummularia Wight & Arn. Saudi J Biol Sci 27:318‒323

Awan SI, SD Ahmad, MA Ali, MS Ahmed, A Rao (2015). Use of multivariate analysis in determining characteristics for grain yield selection in wheat. Sarhad J Agric 31:139‒150

Azeem F, B Ahmad, RM Atif, MA Ali, H Nadeem, S Hussain, H Manzoor, M Azeem, M Afzal (2018). Genome-wide analysis of potassium transport-related genes in chickpea (Cicer arietinum L.) and their role in abiotic stress responses. Plant Mol Biol Rep 36:451‒468

Badouin H, J Gouzy, CJ Grassa, NB Langlade (2017). The sunflower genome provides insights into oil metabolism, flowering and asterid evolution. Nature 546:148‒152

Bari A, M Farooq, A Hussain, M Tahir ul Qamar, MW Abbas, G Mustafa, A Karim, I Ahmed, T Hussain (2018). Genome-wide bioinformatics analysis of aquaporin gene family in maize (Zea mays L.). J Phylogenet Evol Biol 6; Article 1000197

Bi H, N Kovalchuk, P Langridge, PJ Tricker, S Lopato, N Borisjuk (2017). The impact of drought on wheat leaf cuticle properties. BMC Plant Biol 17; Article 85

Blackman BK, M Scascitelli, NC Kane, HH Luton, DA Rasmussen, RA Bye, DL Lentz, LH Rieseberg (2011). Sunflower domestication alleles support single domestication center in eastern North America. Proc Natl Acad Sci USA 108:14360‒14365

Browse J, C Somerville (1991). Glycerolipid synthesis: Biochemistry and regulation. Plant Mol Biol 42:467‒506

Carther KFI, T Ketehouli, N Ye, YH Yang, N Wang, YY Dong, N Yao, XM Liu, WC Liu, XW Li, FW Wang, HY Li (2019). Comprehensive genomic analysis and expression profiling of diacylglycerol kinase (DGK) gene family in Soybean (Glycine max) under Abiotic Stresses. Intl J Mol Sci 20:1361–1379

Chou KC, HB Shen (2010). A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites. PLoS One 5; Article e9931

Fulda M, J Schnurr, A Abbadi, E Heinz, J Browse (2004). Peroxisomal Acyl-CoA synthetase activity is essential for seedling development in Arabidopsis thaliana. Plant Cell 16:394‒405

Gasteiger E, C Hoogland, A Gattiker, S Duvaud, MR Wilkins, RD Appel, A Bairoch (2005). Protein identification and analysis tools on the expasy server. In: The Proteomics Protocols Handbook, pp:571‒607. Humana Press, Totowa, New Jersey, USA

Guo R, L Shi, C Yan, X Zhong, F Gu, Q Liu, X Xia, H Li (2017). Ionomic and metabolic responses to neutral salt or alkaline salt stresses in maize (Zea mays L.) seedlings. BMC Plant Biol 17; Article 41

Islam MA, H Du, J Ning, H Ye, L Xiong (2009). Characterization of Glossy1 homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Mol Biol 70:443‒456

Javed I, S Awan, HM Ahmad, A Rao (2016). Assesment of genetic diversity in wheat synthetic double haploids for yield and drought related traits through factor and cluster analyses. Plant Gene Trait 7:1‒9

Jenks MA, HA Tuttle, SD Eigenbrode, KA Feldmann (1995). Leaf epicuticular waxes of the eceriferum mutants in Arabidopsis. Plant Physiol 108:369‒377

Kunst L, AL Samuels (2003). Biosynthesis and secretion of plant cuticular wax. Progr Lipid Res 42:51‒80

Lai C, L Kunst, R Jetter (2007). Composition of alkyl esters in the cuticular wax on inflorescence stems of Arabidopsis thaliana cer mutants. Plant J 50:189–196

Laila R, AHK Robin, K Yang, JI Park, MC Suh, J Kim, IS Nou (2017). Developmental and genotypic variation in leaf wax content and composition, and in expression of wax biosynthetic genes in Brassica oleracea var. capitata. Front Plant Sci 7; Article 1972

Lü S, T Song, DK Kosma, EP Parsons, O Rowland, MA Jenks (2009). Arabidopsis CER8 encodes long-chain acyl-coa synthetase 1 (LACS1) that has overlapping functions with LACS2 in plant wax and cutin synthesis. Plant J 59:553‒564

Lynch M, JS Conery (2000). The evolutionary fate and consequences of duplicate genes. Science 290:1151‒1155

Pascal S, A Bernard, M Sorel, M Pervent, D Vile, RP Haslam, JA Napier, R Lessire, F Domergue, J Joubès (2013). The Arabidopsis cer26 mutant, like the cer2 mutant, is specifically affected in the very long chain fatty acid elongation process. Plant J 73:733‒46

Pulsifer IP, S Kluge, O Rowland (2012). Arabidopsis long-chain acyl-CoA synthetase 1 (LACS1), LACS2, and LACS3 facilitate fatty acid uptake in yeast. Plant Physiol Biochem 51:31‒39

Rieseberg LH, GJ Seiler (1990). Molecular evidence and the origin and development of the domesticated sunflower (Helianthus annum, Asteraceae). Econ Bot 44:79‒91

Saitou N, M Nei (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406‒425

Samuels L, L Kunst, R Jetter (2008). Sealing plant surfaces: Cuticular wax formation by epidermal cells. Annu Rev Plant Biol 59:683‒707

Schilling EE, CB Heiser (1981). Infrageneric Classification of Helianthus (Compositae). Taxon 30:393‒403

Schnurr J, J Shockey, J Browse (2004). The acyl-CoA synthetase encoded by LACS2 is essential for normal cuticle development in Arabidopsis. Plant Cell 16:629‒642

Seo PJ, SB Lee, MC Suh, MJ Park, YS Go, CMC Park (2011). The MYB96 transcription factor regulates cuticular wax biosynthesis under drought conditions in Arabidopsis. Plant Cell 23:1138‒1152

Shaheenuzzamn M, T Liu, S Shi, H Wu, Z Wang (2019). Research advances on cuticular waxes biosynthesis in crops: A review. Intl J Agric Biol 21:911‒921

Shockey JM, MS Fulda, JA Browse (2002). Arabidopsis contains nine long-chain acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid metabolism. Plant Physiol 129:1710‒1722

Soltis DE, PS Soltis (2003). The role of phylogenetics in comparative genetics. Plant Physiol 132:1790‒1800

Soltis ED, PS Soltis (2000). Contributions of plant molecular systematics to studies of molecular evolution. Plant Mol Biol 42:45‒75

Sun X, T Lei, JB Du, WY Yang (2015). Identification and characterization of two paralogous plastid terminal oxidase genes in soybean. Intl J Agric Biol 17:1275‒1278

Szklarczyk D, A Franceschini, M Kuhn, M Simonovic, A Roth, P Minguez, T Doerks, M Stark, J Muller, P Bork, LJ Jensen, C Mering (2011). The STRING database in 2011: Functional interaction networks of proteins, globally integrated and scored. Nucl Acids Res 39:561‒568

Tamura K, D Peterson, N Peterson, G Stecher, M Nei, S Kumar (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731‒2739

Wang J, DC Jeewani, Z Wang (2019). Composition and morphology of cuticular waxes on the spikes, flag leaf blades and flag leaf sheaths of wheat (Triticum aestivum). Intl J Agric Biol 21:1249‒1255

Waqas M, MT Azhar, IA Rana, F Azeem, RM Atif (2019). Genome-wide identification and expression analyses of WRKY transcription factor family members from chickpea (Cicer arietinum L.) reveal their role in abiotic stress-responses. Genes Genomes 41:467‒481

Yang K, Y Li, S Wang, X Xu, H Sun, H Zhao, X Li, Z Gao (2019). Genome-wide identification and expression analysis of the MYB transcription factor in moso bamboo (Phyllostachys edulis). Peer J 6; Article e6242

Zeisler-Diehl V, Y Müller, L Schreiber (2018). Epicuticular wax on leaf cuticles does not establish the transpiration barrier, which is essentially formed by intracuticular wax. J Plant Physiol 227:66‒74

Zgallai H, K Steppe, R Lemeur (2005). Photosynthetic, physiological and biochemical responses of tomato plants to polyethylene glycol-induced water deficit. J Integr Plant Biol 47:1470‒1478

Zhao Y, X Cheng, X Liu, H Wu, H Bi, H Xu (2018). The wheat MYB transcription factor TaMYB31 is involved in drought stress responses in Arabidopsis. Front Plant Sci 9; Article 1426

Zhou L, E Ni, J Yang, H Zhou, H Liang, J Li, D Jiang, Z Wang, Z Liu, C Zhuang (2013). Rice OsGL1-6 is involved in leaf cuticular wax accumulation and drought resistance. PLoS One 8; e65139

Zhou T, D Yu, H Dong, Z Sun, Y Tan, X Sun, X Sheng, M Duan, DY Yuan (2020). Genome-wide identification and expression profile of NINJA and AFP genes in rice. Intl J Agric Biol 23:171‒182

Zhu L, J Guo, J Zhu, C Zhou (2014). Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis. Plant Physiol Biochem 75:24‒35