**3. Results**

#### **3.1. Sequencing and assembly of** *Persea americana* **var. drymifolia seed transcriptome**

A total of 5005 sequences was assembled and obtained 3328 sequences (171 contigs of 3222 sequences + 106 singletons); these sequences were imported into BLAST analysis. In contig assembly for the avocado seed ESTs, a total number of contigs was 171, derived from 3222 sequences and unique transcripts represented was 106 sequences. Contig length ranged from 84 to 1149 bp (**Figure 1**), the peak EST length was 84–195 bp with 478 sequences in range. The shortest sequence examined was 84 bp to known genes like GTP-binding nuclear protein.

#### **3.2. Functional annotation**

**2.7. Semi-quantitative RT-PCR**

48 Advances in Seed Biology

**Table 1.** Primers used in semi-quantitative PCR.

three repeats.

**3. Results**

The RNA from different seed stages (1, 4 and 8 months of development) digested with DNAse I amplification grade (Invitrogen) was used as a template in sqPCR reactions, and the synthesis of cDNAs was carried out with the First Strand cDNA Synthesis Kit (Thermo Scientific). In Veriti 96-well Thermal cycler (Applied Biosystems), in reactions of

**GenBank Accession Gene name Primer sequence (5′–3′) Tm (°C)** MF353071 PabHLHS1 **F:** TAGCGACAGGATTTGGCAGTTT 65°C

MF353072 PaMT2a **F:** TGAGAGGGAGATTGGAGGAG 54°C

MF353073 PaMT2b **F:** CATGCACCTGCAAATGAGAG 60°C

MF353074 PaMT3 **F:** TTGGTGTTGGTGTCCTATGC 57°C

AGC92009 PaSn **F:** TCCTTGCTTTCCTTCTCTTCAC 64°C

**R:** TGCCCCGTCGTCTCTTTCTTCTACC

**R:** TCCCAACCACAGCATAGTACC

**R:** CACCCAGATACAGCAGGAGAA

**R:** CATGACTCAACCACACACACC

**R:** AGATGTCCCGCAATCTATTTGT SUMO **F:** GATAAGAAGCCCACGGATCA 55°C **R:** GACGGCCATCGAATAAGAAC

polymerase), sqPCR primers (**Table 1**) were designed by Primer3 webtool (http://bioinfo. ut.ee/primer3-0.4.0/); the primers for bHLH transcription factor, three metallothioneins, snakin and ubiquitin as reference gene were selected. The amplification procedures were 95°C - 10min., 30 cycles (95°C - 45 s, annealing 30 s, amplification 72°C - 45 s), 72°C - 7 min. Gene expression ratio between selected gene and endogenous control was calculated using band intensity measured with GelAnalyzer 2010. The semi-quantitative PCR was performed with

**3.1. Sequencing and assembly of** *Persea americana* **var. drymifolia seed transcriptome**

A total of 5005 sequences was assembled and obtained 3328 sequences (171 contigs of 3222 sequences + 106 singletons); these sequences were imported into BLAST analysis. In contig

, 1 U Taq DNA

10 μl (100 ng/μl cDNA,10X Reaction Buffer, 2 mM dNTPs, 50 mM MgCl<sup>2</sup>

Functional interpretation is an important step in the analysis of transcriptomics which cannot be done without the availability of functional annotation. The most widespread and probably most extensive functional annotation schema for gene and protein sequences is the Gene Ontology (GO) [24] as standard in all public databases. Automatic functional annotation methods basically rely on sequence, structure, phylogenetic or co-expression relationships between known and novel sequences [25]. A total of 277 uniESTs sequences were manually annotated for a closer understanding of gene expression in avocado seed. The annotation proceeds through three basic steps: homologs search, GO term mapping and actual annotation. At the first step, NCBI-BLASTX and BLASTN are typically used, and for this work, the e-value 1xE−5, cut-off:33 and the number of 20 retrieved BLAST hits are used. These uniESTs were classified into five functional categories, including antioxidative protection (677, 20.34%), transcription regulatory (1013, 30.44%), defense (507, 15.23%), cellular structure and organization (287, 8.62%) and unknown (844, 25.36%).

**Figure 1.** Sequence length distribution of ESTs from avocado native mexicano seed transcriptome. Total number of sequences analyzed were 3328. Data include both contigs and singletons.

Gene ontology annotations and functional analyses of avocado seed transcriptome were carried out with automated software Blast2GO. These were assigned into three standard classifications: biological processes, molecular functions and cellular components, and summarized according to GO criteria. The majority GO annotation was for biological process (65.34%), cellular component (19.34%) and molecular function (15.33%). In addition, the organisms closely related to the genetic load on avocado seed were reviewed within the databases, and the majority of analyzed sequences (23.47%) (**Figure 2**) were closely related to *Nelumbo nucifera* sequences (**Figure 3**). The 20 most abundant uniESTs and their annotations are shown in **Table 2**.

#### **3.3. Avocado seed abundant genes and validation**

The most abundant sequences match with metallothionein genes. This result suggests that metallothionein genes dominate the avocado seed transcriptome like avocado fruit [26]. Metallothioneins (MTs) were discovered by Margoshes and Vallee as cadmium-bound proteins isolated from the cortex of the equine kidney. These proteins were named for the high sulfur content and metals they are able to bind; depending on the metal species, these may possess more than 20% of its nature of metal ions [27, 28]. Mammalian metallothioneins are 60 amino acid peptides with 20 Cys residues and a molecular mass of about 6–7 kDa. Mammalian MTs are capable of binding up to 7 divalent metal ions via mercaptide bonds (sulfur-metal) with the Cys residues. By convention, any peptide or protein that resembles several characteristics of mammalian metallothioneins can be classified as metallothionein [28]. Plant metallothioneins have two (highly conserved) sequence similarity regions corresponding to the two Cys-rich terminal domains joined by a less conserved "spacer" (about 40 aa without Cys residues). In plants, the most distinctive feature is to have a large spacer, which differs from the MT of the

**Figure 2.** Gene ontology classification of *P. americana* var. drymifolia seed transcriptome. Unigenes with BLASTX matches were classified into three main GO categories: biological process, cellular components and molecular functions.

Transcriptome (ESTs) of Avocado "Native" Mexicano Early Seed Development Shows Abundance... http://dx.doi.org/10.5772/intechopen.70573 51

**Figure 3.** Main organisms with which the avocado seed transcriptome is closely related.

Gene ontology annotations and functional analyses of avocado seed transcriptome were carried out with automated software Blast2GO. These were assigned into three standard classifications: biological processes, molecular functions and cellular components, and summarized according to GO criteria. The majority GO annotation was for biological process (65.34%), cellular component (19.34%) and molecular function (15.33%). In addition, the organisms closely related to the genetic load on avocado seed were reviewed within the databases, and the majority of analyzed sequences (23.47%) (**Figure 2**) were closely related to *Nelumbo nucifera* sequences (**Figure 3**). The 20 most abundant uniESTs and their annotations

The most abundant sequences match with metallothionein genes. This result suggests that metallothionein genes dominate the avocado seed transcriptome like avocado fruit [26]. Metallothioneins (MTs) were discovered by Margoshes and Vallee as cadmium-bound proteins isolated from the cortex of the equine kidney. These proteins were named for the high sulfur content and metals they are able to bind; depending on the metal species, these may possess more than 20% of its nature of metal ions [27, 28]. Mammalian metallothioneins are 60 amino acid peptides with 20 Cys residues and a molecular mass of about 6–7 kDa. Mammalian MTs are capable of binding up to 7 divalent metal ions via mercaptide bonds (sulfur-metal) with the Cys residues. By convention, any peptide or protein that resembles several characteristics of mammalian metallothioneins can be classified as metallothionein [28]. Plant metallothioneins have two (highly conserved) sequence similarity regions corresponding to the two Cys-rich terminal domains joined by a less conserved "spacer" (about 40 aa without Cys residues). In plants, the most distinctive feature is to have a large spacer, which differs from the MT of the

**Figure 2.** Gene ontology classification of *P. americana* var. drymifolia seed transcriptome. Unigenes with BLASTX matches were classified into three main GO categories: biological process, cellular components and molecular functions.

are shown in **Table 2**.

50 Advances in Seed Biology

**3.3. Avocado seed abundant genes and validation**

animals in which the Cys-rich domains are separated by a short spacer of less than 10 amino acids which do not include aromatic residues. The distribution of Cys residues as well as the length of the spacer region served to classify more MT of plants into four types, namely group 1, 2, 3 and 4 [29].

From the analysis of these abundant transcripts, we founded the existence of three MT's genes on avocado seed: PaMT2a, PaMT2b and PaMT3, which were registered in GenBank database with an accession code shown in **Table 3**. Characteristics predicted in silico for avocado metallothioneins, we have two sequences belonging to Methallotionein-2 superfamily due to the two highly conserved Cys-rich motifs and the long spacer in the middle of them. PaMT3 keeps the spacer but Cys-rich motifs are not so conserved grouping this as part of the third family of plant metallothioneins (MT3). Alignment shown that the most conserved amino acids are around the Cys residues for both MT2 and MT3 groups, which are associated with the "metal binding clusters" (**Figures 4** and **5**). The alignments performed identify the amino and terminal carboxyl regions as having the most conserved Cysteine sequences, which correspond to the metal binding clusters. The intermediate spacer of about 40 amino acids is much more variable but has no cysteines. In mammalian metallothioneins, this spacer is very small (8 amino acids) and has no aromatic amino acids. However, in the family 2 to which the plants belong, we can find conserved tyrosine residues in the spacer, as well as several less conserved phenylalanines present in the metallothionein sequences of types 2 and 3 (**Figures 4** and **5**). Seed metallothioneins correspond with the three reported: NnMT2a, NnMT2b and NnMT3 [30]; the avocado seed metallothioneins are closely related with *Nelumbo nucifera* metallothioneins like 79% for NnMT2a-PaMT2a; 66% NnMT2b-PaMT2b and 71% for NnMT3-PaMT3 **Figure 6**. NnMT2a and NnMT3 were associated with processes of seed germination, tolerance to accelerated aging and salt on Arabidopsis [30]. However, avocado seeds have a low tolerance


**Table 2.** Top 20 most abundant annotated ESTs of avocado nativo mexicano seed transcriptome.

to aging. Basic function of MTs is metal homeostasis and has been reported during biotic and abiotic stress conditions too [31]. Recently, it has been reported that one metallothionein interacts with a cytoskeleton protein in the nucleus of rice cells in response to salt stress [32].

Transcriptome (ESTs) of Avocado "Native" Mexicano Early Seed Development Shows Abundance... http://dx.doi.org/10.5772/intechopen.70573 53


**Table 3.** Characterization of Persea americana var. drymifolia metallothioneins.


**Figure 4.** Alignment of plant metallothioneins of subfamily 2.

**Figure 5.** Alignment of plant metallothioneins of subfamily 3.

to aging. Basic function of MTs is metal homeostasis and has been reported during biotic and abiotic stress conditions too [31]. Recently, it has been reported that one metallothionein interacts with a cytoskeleton protein in the nucleus of rice cells in response to salt

**ESTs Description Length e-value GO IDs Abundance**

arlgES153 Metallothionein type 3 195 4.52E-28 GO:0043167 123 arlgES154 Stress response nst1 696 1.68E-34 GO:0009507; GO:0010207 113 arlgES2 40S ribosomal S29 171 1.77E-32 GO:0003735; GO:0043167;

arlgES6 ras-related RIC2 525 2.61E-118 GO:0007165; GO:0043167;

arlgES7 Metallothionein 2 240 3.91E-30 GO:0043167 77

arlgES10 kDa proline-rich 483 4.35E-25 N/A 69 arlgES155 Early nodulin-93-like 318 8.41E-43 GO:0005575 69

arlgES12 60S ribosomal L24 492 5.32E-63 GO:0003735; GO:0022618;

arlgES13 60S ribosomal L7–2-like 738 1.65E-136 GO:0003735; GO:0005829;

arlgES158 Programmed cell death 4 636 1.00E-80 N/A 52 arlgES16 Type 2 metallothionein 243 5.56E-24 GO:0043167 51 arlgES159 Mitochondrial 178 4.72E-35 GO:0005739 50 arlgES161 Snakin 318 4.00E-57 GO:0009740 48

**Table 2.** Top 20 most abundant annotated ESTs of avocado nativo mexicano seed transcriptome.

942 1.35E-103 GO:0046983 123

GO:0006412

282 4.53E-37 N/A 96

579 5.71E-130 GO:0016192; GO:0005575 90

588 1.02E-132 GO:0044281; GO:0006091;

552 2.83E-19 GO:0005739 66

366 2.97E-55 GO:0022857; GO:0009536 64

GO:0005622

GO:0005829; GO:0005840;

GO:0016491; GO:0043167

GO:0005829; GO:0042254; GO:0005840; GO:0006412

GO:0042254; GO:0005840;

GO:0006412

291 3.63E-61 N/A 54

100

78

74

58

57

arlgES1 Transcription factor bHLH96

52 Advances in Seed Biology

arlgES3 PREDICTED:

arlgES4 Vesicle-associated

uncharacterized protein LOC104611921

uncharacterized protein LOC103701850 isoform X3

membrane 726

arlgES8 Isocitrate dehydrogenase [NADP]

arlgES157 Potassium transporter 12 isoform X1

arlgES15 Ethanolamine utilization eutQ

arlgES156 PREDICTED:

stress [32].

Sequences of related proteins were obtained from Zhou et al. [30] and downloaded from NCBI-GenBank: BAD18376.1 GmMT1 (*Glycine max*); BAD18378.1 VaMT1 (*Vigna angularis*); AAF04584.1 MsMT1 (*Medicago sativa*); BAD18382.1 PsMT1 (*Pisum sativum*); BAD18380.1 VfMT1 (*Vicia faba*); ABL10085.1 LbMT2 (*Limonium bicolor*); BAD18383.1 PsMT2 (*Pisum sativum*); Q39459.2 CaMT2 (*Cicer arietinum*); BAD18379.1 VaMT2 (*Vigna angularis*); ABA08415.1 AhMT2 (*Arachis hypogaea*); AAL76147.1 AtMT2a (*Arabidopsis thaliana*); NP\_195858.1 AtMT2B (*Arabidopsis thaliana*); ABN46987.1 NnMT2a (*Nelumbo nucifera*); ABN46988.1 NnMT2b (*Nelumbo nucifera*); CAA10232.1 FsMT2 (*Fagus sylvatica*); ABR92329.1 SmMT2a (*Salvia miltiorrhiza*); NP\_566509.1 AtMT3 (*Arabidopsis thaliana*); AAS99234.1 NcMT3 (*Noccaea caerulescens*); BAB85599.1 BjMT3 (*Brassica juncea*); ACB10219.2 EgMT3 (*Elaeis guineensis*); ACV51811.1 TaMT3 (*Typha angustifolia*); Q40256.1 MaMT3 (*Musa acuminata*); ABN46986.1 NnMT3 (*Nelumbo* 

**Figure 6.** Phylogenetic analysis of avocado and plant metallothioneins. The evolutionary history was inferred using the neighbor-joining method. The bootstrap consensus tree inferred from 1000 replicates. The evolutionary distances were computed using the JTT matrix-based method. The analysis involved 33 amino acid sequences. All positions with less than 85% site coverage were eliminated.

*nucifera*); ACC77568.1 PjMT3 (*Prosopis juliflora*); CAH59438.1 PmMT3 (*Plantago major*); NP\_181731.2 AtMT4 (*Arabidopsis thaliana*); AAB65792.1 GmMT4 (*Glycine max*); P30570.2 TaMT4 (*Triticum aestivum*); AAS78805.1 OsMT4 (*Oryza sativa* Japonica Group) and NP\_001105499.1 ZmMT4 (*Zea mays*), [30].

Another abundant transcript is a messenger codified for a transcription factor type bHLH. The basic domain (bHLH) is a highly conserved amino acid motif that defines a group of transcription factors, which was initially described in animals and was soon discovered in all major eukaryotic lineages [33]. Proteins containing a bHLH domain (referred to as bHLH proteins) are involved in a variety of regulatory processes; their functions include the regulation of neurogenesis, myogenesis and the development of the heart in animals [34, 35], control of phosphate uptake and glycolysis in yeast [36] or modulation of secondary metabolism pathways, epidermal differentiation and environmental responses in plants [37]. The bHLH domain consists of two distinct segments composed by 50–60 amino acids, 10–15 mostly basic amino acids form the stretch (basic region) and approximately 40 amino acids form the two amphipathic helices separated by a loop (helix-loop-helix region). The analysis of the structure of bHLH proteins (yeast and mammalian) showed the basic region made in the DNA contact, while the two helices promote the formation of heterodimers between bHLH proteins [35]. These bHLH transcription factors are generally classified into six major groups (FAs) based on their ability to bind to DNA [35, 38, 39]. Most bHLH proteins are classified into group A or B; in group A, it is expected to bind to E-box consensus sequences (CACCTG or CAGCTG), in group B, it is specifically bind to G-Box consensus sequences (CACGTG or CATGTTG) and in group C, bHLH proteins share a PAS domain and bind to the recognized sequences without a need a E-box (ACGTG or GCGTG) sequences. The E group includes bHLH proteins containing a conserved Pro or Gly residue at a key position within the basic region, preferably bind to sequences referred to as N-boxes (CACGCG or CACGAG), and further share an additional WRPW motif. Groups D and F represent particularly proteins which were separated in the basic region. Some group D proteins have been described as being unable to bind to DNA and could form heterodimers that function as negative regulators of bHLH binding to DNA [40]. Group F includes so-called COE proteins. A phylogenetic study indicated that group A contained mammalian bHLH proteins and lacked bHLH plant proteins. The other groups had a mixture of different species and most of the bHLH proteins of plants belonged to group B [41, 42], It has been shown that the bHLH family of proteins in plants is monophyletic and subjected to significant radiation before the evolution of mosses; bHLH groups established in terrestrial plants during the first 400 million years were conserved during the later evolution of plants, although there were many duplications of genes. The transcription factors are very varied since it does not have many amino acids conserved throughout its sequence; nevertheless in the sites of union to the DNA like the case of the bHLH, the great majority of its amino acids is conserved within its main motive. Due to their propensity to form homodimers or heterodimers, bHLH proteins can participate in an extensive set of a combinatorial interactions leading to the regulation of multiple transcriptional programs. The development of fleshy fruits involves complex physiological and biochemical changes. Recent studies have described the involvement of bHLH proteins in the determination of plant organ size. The SPATULA protein was shown to control cotyledon, leaf and petal expansion by affecting cell proliferation in *Arabidopsis thaliana* [43]. Nicolas et al. [44] described a bHLH transcription factor preferentially expressed in grape berry fruit, but is weakly detected in seeds. This gene is involved in cell size determination. Three basic helix-loop-helix transcription factors (bHLH) were also found to be involved in *Arabidopsis* fruit dehiscence process: ALCATRAZ (*ALC*), SPATULA (*SPT*) and INDEHISCENT (IND); they form a regulatory network that orchestrates

*nucifera*); ACC77568.1 PjMT3 (*Prosopis juliflora*); CAH59438.1 PmMT3 (*Plantago major*); NP\_181731.2 AtMT4 (*Arabidopsis thaliana*); AAB65792.1 GmMT4 (*Glycine max*); P30570.2 TaMT4 (*Triticum aestivum*); AAS78805.1 OsMT4 (*Oryza sativa* Japonica Group) and NP\_001105499.1

**Figure 6.** Phylogenetic analysis of avocado and plant metallothioneins. The evolutionary history was inferred using the neighbor-joining method. The bootstrap consensus tree inferred from 1000 replicates. The evolutionary distances were computed using the JTT matrix-based method. The analysis involved 33 amino acid sequences. All positions with less

ZmMT4 (*Zea mays*), [30].

54 Advances in Seed Biology

than 85% site coverage were eliminated.

the differentiation of the valve margin, allowing seed dispersal [45]. A protein Blast was performed and the sequences that were selected for a multiple alignment were the ones that presented greater coverage and identity with the transcription factor bHLH of Mexican native avocado seed. High sequence variability was found for the nearest bHLH motif, which presenting a large number of conserved amino acids **(Figure 7)**.

However, since they do not have any additional information regarding the function and/or tissue in which the function is performed, we analyzed our sequence with bHLH sequences of which their function or organospecificity is known; two bHLH factors were chosen with these characteristics of *Arabidopsis thaliana*, bHLH Zoupi (GenBank Accession: OAP16519) involved in seed development and SPATULA (GenBank Accession: AT4G36930) involved in

**Figure 7.** bHLH (PabHLHS1) avocado seed transcription factor alignment with bHLH transcription factors of some plant species. *Arabidopsis thaliana* (Ath96bHLH), *Vitis vinifera* (Vvi96bHLH), *Glicine max* (Glm96Bhlh), *Nelumbo nucifera* (96bHLH), *Theobroma cacao* (ThcbHLH), in shadow black are the bHLH motif represented in black letters the amino acids that are conserved in these proteins and gray letters the amino acids differs between them.

cell development; however, no conserved amino acids were found with the avocado bHLH (data not shown). To try to infer the function of this transcription factor highly expressed in avocado seed, it will be necessary to carry out research studies of the recognition boxes inside in DNA to infer the possible association to the group to which it belongs. It is probable that the bHLH transcription factor of the avocado seed has a principal paper in seed differentiation and development.

the differentiation of the valve margin, allowing seed dispersal [45]. A protein Blast was performed and the sequences that were selected for a multiple alignment were the ones that presented greater coverage and identity with the transcription factor bHLH of Mexican native avocado seed. High sequence variability was found for the nearest bHLH motif, which pre-

However, since they do not have any additional information regarding the function and/or tissue in which the function is performed, we analyzed our sequence with bHLH sequences of which their function or organospecificity is known; two bHLH factors were chosen with these characteristics of *Arabidopsis thaliana*, bHLH Zoupi (GenBank Accession: OAP16519) involved in seed development and SPATULA (GenBank Accession: AT4G36930) involved in

**Figure 7.** bHLH (PabHLHS1) avocado seed transcription factor alignment with bHLH transcription factors of some plant species. *Arabidopsis thaliana* (Ath96bHLH), *Vitis vinifera* (Vvi96bHLH), *Glicine max* (Glm96Bhlh), *Nelumbo nucifera* (96bHLH), *Theobroma cacao* (ThcbHLH), in shadow black are the bHLH motif represented in black letters the amino acids

that are conserved in these proteins and gray letters the amino acids differs between them.

senting a large number of conserved amino acids **(Figure 7)**.

56 Advances in Seed Biology

Multicellular organisms produce small cysteinerich antimicrobial peptides (AMPs) as an innate defense against pathogens. Native Mexican avocado seed abundantly express Snakin (PaSn) gene. These kind of AMPs were initially isolated from potato but were later found to be ubiquitous. Novel plant APs isolated include in *Arabidopsis* (family of 12-cysteine peptides).

We identified a single cDNA sequence for snakin/GASA (gibberellic acid-stimulated), which contains a coding sequence of 318 bp and encodes a predicted 106 amino acid peptide. This molecule comprises a 26 amino acid signal peptide (residues 1–26), identified by SignalP (http://www.cbs.dtu.dk/services/SignalP/) and a 79 amino acid mature peptide (**Figure 8**). An amino acid alignment of avocado snakin with other similar APs (**Figure 9**) showed that PaSn has the longest sequence compared with the previously reported StSN1 and StSN2 genes from potato. In addition, PaSn has the 12 characteristic Cys residues of this type of AP. In addition to the highly conserved 12 Cys residues, the other motifs in the PaSn protein consist of residues that are mostly polar, non-polar and basic (**Figure 9**). From these analyses, we hypothesized that the Mexican avocado snakin gene could be involved in plant defense in a similar way to that of the StSN1 and StSN2 genes in potato [7]. Until now, this is the first Snakin gene isolated from a seed.


**Figure 8.** PaSn nucleotide sequence with amino acid frame translation (GenBank: KC012806). ORF in gray shaded; signal peptide (bold-underlined) is followed by the amino acid sequence of the mature peptide; the signal peptide prediction was performed using SignalP program (http://www.Cbs.Dtu.Dk/services/SignalP/).


**Figure 9.** Plant snakin amino acid sequences alignment. Amino acids conserved are indicated by an asterisk in the consensus sequence. The black-shaded cysteine residues are present in all sequences. The sequences included are: Avocado PaSn (KC012806); potato: StSN1 (Q948Z4), StSN2 (Q93X17); *Medicago truncatula:* MtGBRP1 (XP\_003603759), MtGBRP2 (XP\_003589486); *Capsicum annum:* CaSN1 (ACC91329); *Jatropha curcas:* JcGAST1 (ACV70139), JcLTRCOR12 (ACU30848); *Solanum lycopersicum:* SlSNP1 (ADR32106) and *Fragaria x ananassa:* FaGAST1 (AAB97006).

#### **3.4. Expression patterns of selected genes measured by sqPCR**

Expression patterns of five genes from the seed library were studied by semi-quantitative PCR (**Figure 10**); bHLH transcription factor, three metallothioneins, antimicrobial peptide snakin and SUMO like reference gene during avocado seed development. These genes could be divided into three stages according to the time of growth of the seed in avocado fruit. The bHLH gene has an expression pattern comparable to the endogenous gene SUMO (Ubiquitin), suggesting a role throughout the formation and development of the avocado fruit seed possibly modulating the biogenesis of the seed or embryo; since from the first month of formation (E1) to ripening (E8), similar expression levels were present. For the Metallothionein gene group, PaMT3 presented a pattern of constant expression in the three stages of seed

**Figure 10.** Transcript profiling of PaMT2a, PaMT2b, PaMT3, PaSn and PabHLHS1 during seed development. (A) Total RNA was extracted from avocado seeds of 1, 4 and 8 months of development. cDNAs were synthetized and used for sqPCR , the normalizing gene was avocado ubiquitin SUMO. (B) The amount of mRNA of different genes is expressed as the ratio of the densitometric measurement of the sample RT-PCR product to the SUMO corresponding product.

development used slightly above at the peak of expression compared to the endogenous gene but not for PaMT2a which has an initial level of expression low in first month of development, having its maximum expression peak in the stage of 4 months; PaMT2b has its maximum expression peak at the beginning of the fruit formation in the first month, decaying this by month 4 and recovering expression levels for ripening; it should be noted that metallothioneins have been directly involved within various roles within the functions. Some of them as carriers or facilitators of metal ions for processes of defense, synthesis or hydrolysis of reserve components to make them more bioavailable [46]; however, the authors do not reach an agreement to say that the different types or families of metallothioneins play a specific role. The snakin gene has a similar behavior throughout the development of the fruit emphasizing its role within the defense against pathogens or as the first barrier of protection or signaling of attack, making it therefore important to maintain the levels of expression throughout development and possibly after this for fruit protection (**Figure 10**). The expression patterns of the selected genes identified by sqPCR and the different expression patterns of avocado seed transcriptome suggested various roles of these genes in response to seed development and protection in avocado fruit.
