Transcriptomic analysis of pepper roots (Capsicum annuum) under phosphorus deficiency

doi:10.21203/rs.3.rs-1859606/v1

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Transcriptomic analysis of pepper roots (Capsicum annuum) under phosphorus deficiency

https://doi.org/10.21203/rs.3.rs-1859606/v1

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Background: Phosphorus (P) is an important nutritional element needed by plants. Roots obtain P as inorganic phosphate (Pi), mostly in H₂PO^-₄form_.It is vital to plants a sufficient supply of Pi since it participates in important processes like photosynthesis, energy transfer, protein activation, amongst others. The physicochemical properties and the adsorption of Pi to organic material usually makes the bioavailability in soil low causing that crops and undomesticated plants experience variations in accessibility or even a persistent phosphate limitation. There is little information on the influence of Pi availability in an important economic crop such as pepper.

Methods and results: In this study, transcriptome data from pepper roots under low-Pi stress was analyzed in order to identify Pi starvation-responsive genes and their relationship with metabolic pathways and functions. An overall of 62 differentially expressed genes (DEGs) were identified; gene ontology analysis showed DEGs classification into 48 functional categories demonstrating the diversity of genes responding to the stress. The most relevant down regulated DEGs were related to rhythmic process, catalytic activity, and cellular anatomical entity; whereas the up-regulated DEGs were associated to cellular process, biological regulation, metabolic process and response to stimulus categories. KEGG enrichment revealed that metabolic pathways such as porphyrin and chlorophyll metabolism was down-regulated, and galactose metabolism and fatty acid metabolism were up-regulated.

Conclusion: Present results indicate that bell pepper follows diverse processes related to low Pi tolerance regulation, such as remobilization of internal Pi, alternative metabolic pathways to generate energy and regulators of root development.

bell pepper

differentially expressed genes

phosphorus deficiency

RNA-Seq

transcriptome

Plants, being sessile organisms, have limited their range of obtaining nutrients to the place where they are found. The nutritional elements that plants need are diverse, classified into macro and micronutrients based on the concentrations necessary for their survival. The nutritional elements are called macronutrients if plants require a concentration > 0.1% of dry mass [1]. Phosphorus (P) is necessary for plant development and is one of six essential macronutrients (N, P, K, Ca, Mg, and S). P is acquired by plant roots as inorganic phosphate (Pi), mostly in the form of H₂PO⁻ ₄ [2]. An adequate supply of Pi is essential to plants since it is not only a key component of cell molecules such as nucleic acids, ATP, and phospholipids, but it is also a crucial regulator in many cellular processes, including energy production, protein activation, and carbon and amino acid metabolism. [2]. Also, Pi or its organic derivatives activates significant biochemical processes such as photosynthesis and respiration. [3].

However, this important nutrient is unavailable because it establishes unions with cations, especially aluminum and iron under acid soil conditions, and magnesium and calcium under basic conditions. Phosphorus availability is also influenced by ionic strength, concentrations of P and metals, and the existence of competing anions, including organic acids usually making the bioavailability in soil scarce. Both crops and wild plants cope with variation in availability or even a persistent phosphate restriction [2]. In this context, phosphorus bioavailability is considered one significant limitation for crop production [3]. Interestingly, over time plants have developed adaptive responses to enhance their well-being during fluctuations in phosphorus reserve [2]. These adjustments include improved topsoil searching by modifying root architecture and growth, through increased root-to-shoot ratio, increased number and length of root hair; improvement in phosphorus (P) uptake by major activity and number of P-transporters; augmentation of P-recycling and scavenging for phosphate from intracellular and extracellular organic pools; and enhancement of P-economy in metabolic pathway [4–5]. Phosphate deprivation inducible responses are initiated through regulatory pathways involving transcriptional, post-transcriptional, and post-translational regulators by restricting Pi supply. Regulatory pathways arrange the adaptive modifications needed to improve plant condition during variable Pi-supply [5]. Recently, 28 phosphate transporters were identified in Capsicum annuum, most of them were expressed under P-stress, indicating a potential role in Pi mobilization; also, they had expression in diverse tissues like root, stem, leaf, flower and fruits, which suggests possible participation in growth and development processes [6]. RNA-sequencing has become an effective tool for the analysis of plant genome expression profiles under a diversity of environmental and developmental circumstances [4]. In recent years, assays related to transcriptomic analysis of the molecular responses to Pi deficiency have been performed in many species, such as rice, common beans, white lupine, and maize [2, 4, 7–8]. To date, there are very few reports on phosphorus deficiency in a crop of economic importance such as pepper; since it is used in different cuisines around the world and also has nutraceutical potential.

In this study, the transcriptome data from pepper roots (Capsicum annuum, L.) was analyzed in order to identify Pi starvation-responsive genes and their relationship with diverse metabolic pathways and functions. Present results will help to a greater understanding of the molecular mechanisms in bell pepper roots during Pi deficiency.

Plant material and growth conditions

The experiments were conducted using bell pepper plants (Capsicum annuum, L.) cv. Cannon (Syngenta). After germination in trays the seedlings were transferred to a hydroponic system with coconut fiber substrate once the third true leaf emerged. The composition of the nutrient solution was as follows: 4 mmol L^− 1 Ca (NO₃)₂ · 4H₂O, 6 mmol L^− 1 KNO₃, 1 mmol L^− 1 KH₂PO₄, 2 mmol L^− 1 MgSO₄ · 7H₂O, 100 µmol L^− 1 Na · Fe EDTA, 46 µmol L^− 1 H₃BO₃, 9.146 µmol L^− 1 MnCl₂ · 4H₂O, 0.76 µmol L^− 1 ZnSO₄ 7H₂O, 0.32 µmol L^− 1 CuSO₄ · 5H₂O, 0.0161µmol L^− 1 (NH₄) 6Mo₇O₂₄ · 4H₂O, pH 6.0 [9–10]. After 3 days of adaptation, five plants per sample were randomly selected and fertilized with phosphorus-deficient nutrition (1 µM P). After applying phosphorus-deficient nutrition (taking as time 0), the root tissue was collected after 3 h, plants with P-sufficiency were taken as the control. The collected tissues were immediately frozen in liquid nitrogen and stored at -80° C for later use [11].

RNA extraction and RNA-sequencing

For this assay, roots of five bell pepper plants with phosphorus deficiency and sufficiency were used for total RNA extraction using the TRIzol® methodology (Life Technologies); each sample had a replica. Total RNA with absorbance ratios of A₂₆₀/A₂₈₀ ≥ 1.8, and A₂₆₀/A₂₃₀ ≥ 1, and RIN values (RNA Integrity Number) ≥ 8 were selected for cDNA libraries synthesis of 150 paired-end reads in LANGEBIO (Irapuato, Mexico). According to the sequencing provider, the libraries were sequenced using the Illumina NextSeq-500 platform.

Bioinformatic data analysis

Once the transcriptome sequences were obtained, the raw reads quality was visualized using the FastQC tool on the galaxy platform (https://usegalaxy.org/). Then, raw reads including the adaptor sequences and low-quality sequences were filtered into clean reads using Trimmomatic (https://usegalaxy.org/) with the following parameters: ILLUMINACLIP using the adapter sequence option of Truseq3 (additional seqs), a quality score of 20 (SLIDINGWINDOW:4:20) and minimum reading length of 35 (MINLEN:35). Clean reads were aligned to Capsicum annuum cv. Zunla-1 genome reference by hierarchical indexing using HISAT2 [12–13]. The quantification of mapped reads per gene was performed using HTSeq-count [14], and the differentially expressed genes (DEGs) among treatments were identified by DESeq2. This analysis is based on a model using the negative binomial distribution. The false detection ratio (FDR) was set to α ≤ 0.05 according to the criteria of a Log2-FC in expression and a p-adjusted value < 0.05 [15–16].

Gene ontology and KEGG enrichment analysis

The GO enrichment of DEGs was performed in PANTHER (http://www.pantherdb.org/) web-based tool for GO analysis. GO terms were performed with FDR ≤ 0.05. We carried out the statistical enrichment of the differential expression genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) in order to classify DEGs to specific biological pathways (https://www.kegg.jp/), p-adjusted value ≤ 0.05. DEGs were also mapped to gene ontology (GO) terms in the database (http://www.geneontology.org/).

Real-time PCR analysis of candidate genes

Total RNA was isolated from pepper roots exposed to low phosphorus with 0 h (control) and 3 h, following the TRIzol methodology (TRIzol® Reagent from Life technologies). Total RNAs from each time point were purified and then used for cDNA synthesis by RT-PCR. It was carried out from 1 µg of RNA, using the commercial kit Super Script III first strand synthesis for RT-PCR (Invitrogen) according to the manufacturer's procedure. For the qPCR of the candidate genes the PCR program was a cycle of 5 min at 97°C, 40 cycles of 15s at 95°C and 15 s at 60°C, 1 min at 60°C and 15 s at 95°C. To estimate the relative expression, we used the 2^−ΔΔCt formula (Livak and Schmittgen, 2001; Romero et al., 2015).

Data Processing

A total of 157,323,048 paired-end raw reads were obtained in this study, with an average of 19,665,381 reads per sample. After filtering with Phred score ≥ 20 and minimal length > 35 bp, preserving an average of 79% of the total raw reads (Table S1). Read alignments had an overall alignment between 50.37% and 80.53% of the filtered reads (Table S2).

Expression profile of differentially expressed genes in pepper roots

Genes considerably up-regulated and down-regulated by Pi-deficient conditions were identified using the statistical package DESeq2 (Table S3). After 3 h of low phosphorus stress in pepper roots, a total of 62 genes were differentially expressed, of which 56 genes were up-regulated (Table 1) and 6 genes were down-regulated (Table 1) (Fig. 1). Also, cluster analysis showed different transcriptomic profiles between the control and the Pi deficiency samples (Fig. 2).

GO‑term analysis of DEGs

The identified DEGs were aligned with the GO database in order to be classified into functional categories in this study. Twenty-six GO categories were identified for the down-regulated genes (Fig. S1). Of these, 3 were related to cellular component (CC), 5 GO terms were associated to molecular function (MF), and 18 GO terms were related to biological process (BP). In the MF category, catalytic activity and union were the prevailing GO terms, while the predominant GO terms for the BP category were cellular process, metabolic process, and reproduction. Cellular anatomical entity, intracellular and protein-containing complex were the GO terms associated with CC. On the other hand, 22 GO categories were identified for the up-regulated genes (Fig. S2), in which cellular process, metabolic process and response to stimulus were the leading GO terms between the 11 terms in the BP category. Regarding the CC category, 3 GO terms were identified, including intracellular, protein-containing complex and cellular anatomical entity. Finally, in the MF category, 8 GO terms were determined, catalytic activity and binding were the prevailing GO terms. These results suggest that low Pi treatment may lead to different responses from the plant in order to counteract the effects of the deficiency.

KEGG pathways analysis

To determine the biological functions of the DEGs, the differentially expressed genes were mapped to the KEGG database. A total of 92 metabolic pathways were identified in which these genes were involved (Table S4, Table S5); the top 10 up-regulated pathways are presented in Fig. 3a according to the enrichment significance. The top 10 down-regulated are also revealed in Fig. 3b. Pathways with a p-value ≤ 0.05 were considered significantly enriched pathways. The most important were those related to “Porphyrin and chlorophyll metabolism” (cann00860), “Circadian rhythm-plant” (cann04712) (Table S5), “Protein processing in endoplasmic reticulum” (cann04141), “Galactose metabolism” (cann00052), “Endocytosis” (cann04144), “Biosynthesis of unsaturated fatty acids” (cann01040) and “Fatty acid metabolism” (cann01212) (Table S4). These results showed that genes involved in these biological pathways dramatically changed gene expression levels in response to phosphorus deficiency stress. Moreover, from these transcriptomic data, more in-depth research was carried out in order to understand the mechanisms of regulation of certain pathways in bell pepper roots under low Pi.

Differentially expressed genes associated to metabolism

Throughout the 3 h of Pi starvation, some metabolic responses were initiated. It was expected to see genes related to plant growth as a metabolic adaptation (down-regulation of proteins synthesis and up-regulation of protein degradation). There was one down-regulated gene and 13 up-regulated genes associated with the metabolism of cofactors and vitamins (porphyrin and chlorophyll). These genes were mainly involved in protein processing, secondary metabolic pathway genes, fatty acid degradation and carbohydrate metabolism. From these, 17.8 kDa class I heat shock protein, heat shock cognate 70 kDa protein 2-like, heat shock cognate 70 kDa protein 1, heat shock protein 83-like, E3 ubiquitin-protein ligase RNF181-like, small heat shock protein chloroplastic-like, and small heat shock protein (chloroplastic) were involved in protein processing. In addition, alcohol dehydrogenase 1, UTP-glucose-1-phosphate uridylyltransferase-like, 1-aminocyclopropane-1-carboxylate oxidase 1 are involved in biosynthesis of secondary metabolites; phosphatidylinositol 4-phosphate 5-kinase 9-like takes part in carbohydrate metabolism and alcohol dehydrogenase 1 in fatty acid degradation. Also, thiamine thiazole synthase 1 (chloroplastic) and ferredoxin (root) R-B2-like are involved in vitamin and cofactors and energy metabolism, respectively.

Table 1

Up-regulated and down-regulated genes in response to Pi deficiency in pepper roots.
Gene_ID	Gene_symbol	Log2_FC
LOC107858740	cyclic dof factor 3-like	-1.62803167
LOC107870073	magnesium-chelatase subunit ChlH, chloroplastic	-1.28552323
LOC107839902	protein EXORDIUM-like 5	-1.24458076
LOC107842877	ribulose bisphosphate carboxylase/oxygenase activase 1, chloroplastic	-1.05172981
LOC107870484	protein RSI-1	-1.03438469
LOC107864326	glycine-rich RNA-binding protein 2, mitochondrial	-1.01357798
LOC107871185	Uncharacterized	1.0010666
LOC107877758	hemoglobin-2	1.00245233
LOC107877102	putative elongation of fatty acids protein	1.00417779
LOC107867437	protein NRT1/ PTR FAMILY 6.3-like	1.00463201
LOC107860678	Titin	1.01398858
LOC107840006	actin-100	1.02361085
LOC107842555	probable glutathione peroxidase 5	1.0337885
LOC107875359	peptidyl-prolyl cis-trans isomerase FKBP62	1.03758765
LOC107843330	phosphatidylinositol 4-phosphate 5-kinase 9-like	1.04139975
LOC107871161	alkaline/neutral invertase A, mitochondrial-like	1.04570789
LOC107843583	Uncharacterized	1.06333511
LOC107857067	transcription factor HBP-1b(c38)-like	1.08520371
LOC107875506	17.8 kDa class I heat shock protein	1.08725969
LOC107870215	heat shock cognate 70 kDa protein 2-like	1.10658529
LOC107856341	thiamine thiazole synthase 1, chloroplastic	1.11084087
LOC107847569	dnaJ protein homolog	1.11130542
LOC107843077	aquaporin PIP2-1-like	1.11141118
LOC107867171	ethylene-responsive transcription factor ERF010-like	1.12046085
LOC107853861	multiprotein-bridging factor 1c	1.1290481
LOC107840522	uncharacterized protein	1.15031467
LOC107878806	nuclear transcription factor Y subunit B-3-like	1.15366804
LOC107839827	protein NRT1/ PTR FAMILY 4.3	1.1547118
LOC107879981	Uncharacterized	1.15517825
LOC107851664	ferredoxin, root R-B2-like	1.16550818
LOC107873027	alcohol dehydrogenase 1	1.16692881
LOC107875543	heat shock cognate 70 kDa protein 1	1.17143161
LOC107864060	ethylene-responsive transcription factor ERF071-like	1.18150539
LOC107871190	probable aquaporin PIP1-2	1.2105557
LOC107872944	probable aquaporin TIP-type RB7-5A	1.2177903
LOC107843566	aquaporin PIP2-2-like	1.21886322
LOC107853722	UTP–glucose-1-phosphate uridylyltransferase-like	1.23150321
LOC107843078	aquaporin PIP2-1-like	1.23243473
LOC107863207	heat shock protein 83-like	1.24270078
LOC107863712	uncharacterized LOC107863712	1.25206873
LOC107853001	uncharacterized acetyltransferase At3g50280-like	1.25959391
LOC107839683	universal stress protein A-like protein	1.27058557
LOC107839668	dnaJ homolog subfamily B member 6-B	1.2715932
LOC107861646	dnaJ homolog subfamily B member 6	1.30508232
LOC107875496	serine/arginine-rich splicing factor SR45a-like	1.30863826
LOC107861978	probable WRKY transcription factor 40	1.31004422
LOC107868099	E3 ubiquitin-protein ligase RNF181-like	1.31442988
LOC107877488	zinc finger protein CONSTANS-LIKE 5	1.32310404
LOC107863805	heat stress transcription factor A-7a-like	1.3364313
LOC107861213	Uncharacterized	1.35830299
LOC107866988	Uncharacterized	1.36381372
LOC107847728	serine carboxypeptidase-like	1.36839351
LOC107862631	BTB/POZ and TAZ domain-containing protein 1-like	1.37549771
LOC107855040	ethylene-responsive transcription factor ERF054	1.44037069
LOC107867615	small heat shock protein, chloroplastic-like	1.57174411
LOC107867608	small heat shock protein, chloroplastic	1.61128536
LOC107850880	1-aminocyclopropane-1-carboxylate oxidase 1	1.67891355
LOC107838978	heat shock factor protein HSF30	1.78439963
LOC107842826	ultraviolet-B receptor UVR8-like	2.0134887
LOC107840613	phosphoprotein ECPP44	2.02241417
LOC107855063	protein GIGANTEA-like	2.12899223
LOC107850365	protein GIGANTEA-like	2.36218548

DEGs related to transcription regulation

Fourteen transcription factor genes were differentially expressed, the expression of 13 of them was up-regulated, and one was down-regulated (Table 1) under Pi-starvation. Up-regulated genes, ethylene-responsive transcription factor ERF071-like, ethylene-responsive transcription factor ERF054, ethylene-responsive transcription factor ERF010-like are related to root development. BTB/POZ and TAZ domain-containing protein 1-like were also identified, which might be implicated in Pi metabolism by transcription factor (TF) interaction. Also, dnaJ homolog subfamily B member 6-B, dnaJ homolog subfamily B member 6, probable WRKY transcription factor 40, heat stress transcription factor A-7a-like, heat shock factor protein HSF30, transcription factor HBP-1b(c38)-like, multiprotein-bridging factor 1c, nuclear transcription factor Y subunit B-3-like related to stress response were identified. Finally, the cyclic dof factor 3-like was down-regulated which is related to developmental processes like flowering time.

DEGs related to transportation

Genes related to transportation are involved in a massive number of vital processes in plants, including the transport of macro and micro-molecules. Seven DEGs encoding transport proteins were found up-regulated in this study. Among these, protein NRT1/ PTR FAMILY 6.3-like, phosphatidylinositol 4-phosphate 5-kinase 9-like, and protein NRT1/ PTR FAMILY 4.3 are related to phosphate transportation. And the rest of them, probable aquaporin PIP1-2, probable aquaporin TIP-type RB7-5A, aquaporin PIP2-2-like, and aquaporin PIP2-1-like, are related to signaling and cellular processes by transporters.

DEGs related to stress response

Several genes were relevant in stress response, it was observed the down regulation of the gene protein RSI-1, which is related with response to salicylic acid, and plant growth, regulation, development, and ripening, it is also related to heat acclimation. In relation with the up-regulated genes, a glutathione peroxidase 5 was detected and could be associated with oxidative stress response, it may constitute a glutathione peroxidase-like protective system, 17.8 kDa class I heat shock protein participates as chaperones and folding catalysts usually in response to exogenous stress. The genes heat shock cognate 70 kDa protein 2-like and heat shock cognate 70 kDa protein 1 are phosphoprotein phosphatases (PP5)-interacting proteins. Alcohol dehydrogenase 1 participates in numerous pathways to generate energy and responds to ABA, which associates this gene with plant development and root growth. Concerning to signal transduction mechanisms, transporters and response to stress are the probable aquaporin PIP1-2, probable aquaporin TIP-type RB7-5A, aquaporin PIP2-2-like, aquaporin PIP2-1-like, universal stress protein A-like protein and E3 ubiquitin-protein ligase RNF181-like.

Differentially expressed genes in pepper roots have different functions in response to low Pi

Nowadays, the absorption capacity of nutrients by plants has become an important topic of different investigations, being the modifications in the architecture of the root part of it [17]. Therefore, studies have emerged to understand the molecular mechanisms that arise in plants exposed to nutrient deficiency [11, 9, 18]. A feature previously reported in Arabidopsis thaliana plants is the change in number and density of lateral roots [11]. This is because, as a measure of adaptation to this nutritional deficiency, the plant generates a larger absorption zone in the soil to capture the available phosphorus [19]. Therefore, the regulation of the root architecture establishes an important point of the adaptative response. In this study, the differential expression of 62 genes was identified, including those that encode the transcription factors ERF071 and ERF010, which regulate genes related to the signaling cascade via ethylene pathways, a phytohormone associated with the regulation of root growth and the formation of root hairs [20]. It has also been observed that ethylene participates in the regulation of the primary root growth under conditions of phosphorus deficiency, which is important for plants since the root provides support to the stem and determines the growth of the plant [21]. This phytohormone interacts with the signaling pathway by auxins, where it has been observed that it induces the growth of lateral roots by mediating the production of auxins and, therefore, the activation of signaling pathways related to this molecule [22].

Also, the up-regulation of the probable transcription factor WRKY 40 and the down-regulation of magnesium chelatase subunit ChlH was observed. ChlH magnesium chelatase subunit encodes a protein with multiple functions. Still, it is mainly implicated in the regulation of protein synthesis related to photosynthesis and with the perception of abscisic acid (ABA). While the transcription factor WRKY 40 acts as a negative regulator on ABA-mediated signaling, suggesting a decrease in energy generation capacity of the plant [23].

Biological pathways involved with stress response in pepper roots under low Pi

Endocytosis is reported to mediate nutrient uptake, receptor internalization, and regulation of cell signaling. The genes identified in this pathway are PPK (phosphatidylinositol phosphate kinase) related to the development of root hairs and a heat shock protein. The first has been reported to target the endosomal localization of regulatory proteins by binding to PX domains, while the second is a protein that is expressed under stress conditions to protect cells and that has also been reported to interact with aquaporins (AQPs) to regulate acquisition of nutrients. Since plants do not have a specialized circulatory system, they rely on AQPs to transport solvents, selected solutes and even reactive oxygen species (ROS); therefore, these proteins have a huge impact during abiotic stress response in plants. Pathways such as glycolysis and gluconeogenesis were also identified, whose metabolites have been reported to participate as signal molecules mediating multiple physiological responses to stress [24–25]; also, accumulated sugars in bell pepper roots were identified under chill stress as well as organic acids and other metabolites [26]. It has been reported that the accumulation of major soluble carbohydrates (e.g., glucose, fructose, and maltose) in bell pepper roots is a common response to abiotic stress; since these metabolites in addition to acting as osmoprotectants, helping preserve the osmotic balance and stabilizing macromolecules under stress conditions, they can also be an energy source for plants [26], which in this case of low-Pi induced stress are focusing their machinery on growth processes (remodeling root morphology and architecture) [27]. Genes related to the metabolism of galactose were identified; according to a recent study, the L-galactose pathway is the prevailing mechanism for Ascorbic acid/ascorbate (AsA) synthesis in leaves and fruits of chili pepper [28–29]. It has also been reported that the rise in the expression of genes linked to organic acid metabolism and their exudation under Pi deficiency, suggests that organic acids play a significant part in the adaptation to Pi-limiting conditions [28–30]. It is known that plants have developed different morphological and physiological modifications to improve Pi acquisition under P-limiting conditions, one of them is the increase of organic acid exudation, establishing a symbiotic relationship with arbuscular mycorrhiza or other beneficial microorganisms; related to this, roots also secrete flavonoids to delay the microbial degradation of secreted organic acids and to enable rhizosphere Pi mobilization [29].

Alcohol dehydrogenase 1 participates in the previously mentioned routes as well as in the metabolism of fatty acids, related to processes of generation of energy molecules (NADH+), through these pathways. It also participates in response to the phytohormone ABA that participates in mediating the development processes, including the increase in root to shoot ratio and root hair density. Related to the circadian rhythm, the GIGANTEA protein was found, which is related to the regulation of the flowering cycle, and it has been reported that the overexpression of this gene in Arabidopsis plants cause early flowering; a mechanism that has been seen as adaptive in another type of stress (drought), which is why it is suggested that under phosphorus deficiency the plant is following this survival mechanism. Genes HEAT SHOCK, BTB/POZ, and TAZ 1 type were identified, the first one is related to responses to stress, and the other two elements have been reported to participate in the regulation of transcription and degradation in proteasome, and ubiquitin-protein mediates degradation in proteosomes [24], which suggests that in some way, protein expression is being modulated and folded in order to recycle them to control some essential cellular functions [5, 20, 23].

In the present study, various genes were identified which play an important role in adaptations suffered by the plant to cope with the low availability of phosphorus, including the participation of PIPs and PTRs, which are involved in the homeostasis of the plant and the transportation of Pi and auxins, inducing root formation. In addition, BTB1 acts as a mediator of multiple responses to stress nutrients and auxins. Molecules such as ERF were also found and have been reported to activate the transcription of WRKY and MYB factors, which, together with zinc finger proteins, modulate responses to abiotic stress as well as defense against oxidative stress. The responses of these factors suggest the presence of a complex network of adaptation to a deficiency at the transcriptional level. Regarding the metabolic routes, it is suggested that the plant generates energy through alternative routes to optimize the use of internal Pi. Some of the products of carbon metabolism pathways also play an important role in root growth (laterals, root hairs). In addition, when secreted from the roots, it facilitates the absorption of Pi from the soil, chelating the Pi in the soil or modifying the rhizosphere improving absorption.

Acknowledgments

This research was supported by the funds FOSEC SEP-INVESTIGACIÓN BÁSICA, Proyecto No. A1-S-8466. Cátedras CONACYT: Proyecto No. 784. Lightbourn Research. Convenio: 589683, Proyecto: Análisis Transcripcional de Pimiento Morrón (Capsicum annuum L.) bajo estrés abiótico.

Author contributions

All authors contributed to the study, conception and design. Material preparation, data collection and analysis were performed by [Daizha Salazar], [Claudia Villicaña], and [Josefina León-Félix]. The first draft of the manuscript was written by [Daizha Salazar] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Conflict of interest: The authors declare that they have no conflict of interest.

Research involving human participants and/or animals This research is about transcriptomic analysis in a plant (pepper roots). Human participants/animals were not involved in this study.

Informed consent Not applicable in this study.

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Transcriptomic analysis of pepper roots (Capsicum annuum) under phosphorus deficiency

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