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. 2022 Nov 1;12(12):335. doi: 10.1007/s13205-022-03399-6

Purple acid phosphatases in coffee—genome-wide identification and the transcriptional profile of selected members in roots upon Pi starvation

Diego Júnior Martins Vilela 1,✉, Renan Terassi Pinto 2, Thiago Bérgamo Cardoso 3, Luciano Vilela Paiva 4, Marco Aurélio Carbone Carneiro 5, Gladyston Rodrigues Carvalho 6, Jessé Valentim dos Santos 7
PMCID: PMC9622964  PMID: 36330378

Abstract

Phosphorus (P) availability is determinant for crop productivity and, despite the sufficient amount of this nutrient in arable land, most of it remains insoluble, leading to the need of high fertilizer input. To cope with P scarcity forecasts and also for cropping costs alleviation, genotypes better adapted to promote P solubilization and absorption are required, especially for perennial crops. Coffee is one of these important perennial crops cultivated in soils with low P availability, and thus we aimed to study adaptive strategies to coffee genotypes in acquire phosphorus. In this study, we focused on rhizosphere phosphatase activity, a major characteristic related to P solubilization from organic pools. To explore the genetic basis of this characteristic, we firstly identified 29 Purple Acid Phosphatases (PAP) genes in C. canephora genome and selected five candidates with higher potential to encode secreted PAPs. We found that C. arabica genotypes have diverse profiles of rhizosphere phosphatase activity, as well as microbial biomass carbon, which we measured to explore the impact of the plant on microbiota and also to discriminate the phosphatase activity origin (plant or microorganism-derived). We selected two C. arabica cultivars with contrasting phosphatase activity and found that one PAP gene has a correlated gene expression profile with this characteristic. This work explores coffee adaptative responses to P starvation conditions, and the information provided can further contribute to breeding programs aiming better adapted genotypes for sustainable agriculture in face of current challenges.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-022-03399-6.

Keywords: Coffea arabica, Coffea canephora, Gene expression, PAP, Phosphorus

Introduction

The demand for food on the planet grows sharply, and one of the main strategies to supply the need of agricultural products is to develop productive crops and enable the efficient use of resources, such as fertilizers (Cordell et al. 2009). The improvement on the plant’s ability to uptake the nutrients from the soil is one important trait to be explored, especially in the case of phosphorus, as an essential macronutrient for the growth and development of plants (Marschner and Rimmington 1988; Vance 2001). To cope with low phosphorus availability, plants have developed a wide range of strategies to improve this nutrient use efficiency, such as increased root system area, association with arbuscular mycorrhizal fungi, increased microbial biomass near the rhizosphere and phosphatase release by the root system and the rhizosphere mutualistic microorganisms (Chiou and Lin 2011; Wu et al. 2013; Hur et al. 2010; Liang et al. 2010; Xiao et al. 2006).

Since phosphorus has low mobility in the soil, root development to achieve P is one of the main plants strategies (Aziz et al. 2011). In addition, with the increase in the root system, there is an increase in soil microbial biomass, which is the living part of the soil’s organic matter, composed of all organisms smaller than 5 × 10−3 μm3. They are the main source of enzymes in the soil and responsible for practically all biological activity, participating as a catalyst in the main biochemical transformations in the soil, such as decomposition/mineralization and solubilization, among which phosphatase stands out (Moreira and Siqueira 2006). Besides root growth and mutualistic relations with microorganisms, root exudates play an important role on P starvation response. Among the substances secreted, purple acid phosphatases (PAPs) are proved to influence P acquisition for some species (Zhu et al. 2020; Deng et al. 2020) and also internal homeostasis of this nutrient upon stressful conditions. Genes codifying these proteins are present in families on plant genomes (Zhu et al. 2020; Venkidasamy et al. 2019) and since they can play important role for plant adaptation under P starvation condition, we aimed to identify all the candidate members on the C. canephora genome (Denoeud et al. 2014).

As a perennial crop, coffee plants remain on the field for decades, growing mostly on P poor soils and phosphorus fertilizing is thus required for keeping high yields. Focusing on P efficient cultivars breeding can be part of a strategy to maintain productive plantations with low P input, thus increasing sustainability. For this purpose, the identification and characterization of genes which improve this nutrient uptake, such as PAPs, must be conducted, as they can be used for targeted conventional breeding or applied to genetic engineering strategies (Mehra et al. 2017; Deng et al. 2020).

To provide deeper insight about coffee PAPs, we performed a semi-controlled experiment with C. arabica genotypes with contrasting phenotypes regarding P use efficiency (Vilela et al. 2021). We analyzed the microbial biomass carbon on their rhizosphere, acid phosphatase activity and then selected two cultivars for gene expression analysis. Five PAPs were selected for RT-qPCR analysis, and we found one gene with expression profile correlated with acid phosphatase activity on coffee rhizosphere, which represents a valuable candidate for further exploration on breeding P efficient cultivars.

Materials and methods

Plant material and experimental design

The in planta experiment was conducted in a greenhouse of the Agricultural Research Corporation of Minas Gerais (EPAMIG), located in Lavras–Minas Gerais, Brazil, at latitude 21°14′30″ south and longitude 45°00′10″ west and altitude of 918.8 m. The average temperature and relative humidity of the greenhouse during the experiment were 23 °C and 65%, respectively.

The properties of the soil used in this experiment are given in Table 2 supplementary. Liming was carried out following the recommendations for coffee crop (Guimarães et al. 1999). After liming, the soil was packed in pots and incubated for 30 days, being moistened to 60% of the field capacity daily.

The coffee seedlings used in the experiments were produced with commercial substrate Plantmax® and transplanted to the pots when they presented three pairs of leaves.

The experimental design was a randomized block, in a 10x2 factorial scheme (10 coffee genotypes and two phosphate fertilizer doses), with four replicates. Each experimental plot consisted of a pot with 10 L of soil, with one plant. The genotypes used were the cultivars Paraíso MG H 419-1, Catuaí Vermelho IAC 144, Catiguá MG2, Topázio MG 1190, Bourbon Amarelo IAC J10, Sarchimor MG 8840, MGS Aranãs, MGS Ametista, MGS Paraíso 2 and the progeny H 6-47-10 pl. 3. The two dosages of phosphate fertilization were zero (without phosphate fertilization) and the dose of 200 mg dm−3 of P per pot. Phosphate fertilization was carried out in a single application, one week before transplanting the seedlings. In addition, each pot received 300 mg dm−3 of N and 200 mg dm−3 of K, divided into five times, applied every 30 days, with the first application made on the date of transplanting the seedlings to the pots. The dosages used were adapted from Novais et al. (1991). All fertilizations were made using nutritive solutions. The pots were irrigated 3–4 times a week, varying with the growth of the plants over the months, and the volume of water added was calculated so that there was no overflow.

The experiment remained for eight months in a greenhouse (November–July). Then, it was disassembled, and samples of soil and plant material (root tips) from each plot were collected for analysis. About 10 root apices of approximately 1 cm were collected in each plot. The collected root samples were immediately immersed in liquid nitrogen and stored at −80 °C for RNA extraction.

Microbiological analyses

The acid phosphatase activity (FFT) was determined according to the methodology described by Tabatabai and Bremner (1969). For the determination of microbial biomass carbon (MBC), was adopted the fumigation–extraction methodology proposed by Vance et al. (1987), obtained by the difference between carbon extracted from fumigated soil and control.

Identification of purple acid phosphatases (PAP) on C. canephora

Amino acid sequences of PAP family members in Arabidopsis thaliana (29 sequences, Li et al. 2002) and Oryza sativa (26 sequences, Zhang et al. 2011) were used as query for local Blastp analysis against the Coffea canephora proteome (Denoeud et al. 2014) downloaded from Coffee Genome Hub (http://www.coffee-genome.org/), with an e-value filter of 0.00001. Unique sequences were filtered from the output file, and these were analyzed for conserved domains common to PAP proteins (such as cl26054 and cl13995), using the NCBI Conserved Domain Search software (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

For the selection of proteins in coffee belonging to the PAP family, the sequences resulting from the previous analysis were analyzed for the presence of specific amino acid sequence patterns for this family (Tian and Liao 2015): DXG, GNH(D/E), GDXXY, VXXH and GHXH. All the proteins were submitted to Expasy (https://www.expasy.org/resources/compute-pi-mw) to obtain information regarding this molecular weight and isoelectric point and, furthermore, the DeepLoc 2.0 (https://services.healthtech.dtu.dk/service.php?DeepLoc-2.0; Thumuluri et al. 2022) software was utilized to predict their subcellular localization (Suppl. Table 1). Then, the proteins selected in coffee were aligned using the ClustalW algorithm (Thompson et al. 1994) and phylogenetically analyzed in association with the proteins of Arabidopsis thaliana, Glycine max and Oryza sativa, using the Neighbor joining method (Saitou and Nei 1987) with a model substitution p-distance (Nei and Kumar 2000) and 1000 bootstrap replicates, via MEGA 7.0 software (Kumar et al. 2016).

RNA purification, cDNA conversion and qPCR

RNA extraction was performed using the ConcertTM Plant RNA Reagent (Invitrogen), following the protocol recommended by the manufacturer. At the end of the extraction, the RNA obtained from each sample was subjected DNAse treatment (Turbo DNA-free Kit, Ambion Biosciences), according to the manufacturer’s methodology.

The purified RNA of each sample was then evaluated for integrity, quality and quantity via agarose gel electrophoresis and spectrophotometric analysis on NanovueTM equipment (GE-Healthcare Life Sciences). Electrophoresis was performed with 1 µL of total RNA in 1% agarose gel at 100 V for 40 min, and only samples with clear, separate and proportional ribosomal RNA bands were considered to be intact (Suppl. figure 1). In the spectrophotometric analysis, samples with a quantity greater than 300 ng µL−1 were selected, with purity measured by analyzing the ratios of RNA compared to total proteins, phenolic compounds and salts via absorbance at 280 and 230 nm wavelengths. The samples that presented values close to the range of 1.8–2.2 for the ratios between these wavelengths and passed through integrity analysis were considered as suitable for RT-qPCR.

The purified RNA samples were submitted to a PCR with primers for amplification of a selected endogenous gene (24s, Suppl. Table 4) (PCR Housekeeping) to verify the absence of remaining DNA. The result was analyzed via electrophoresis on a 2% agarose gel, performed at 100 V for 40 min, where only samples that were not detected in the gel were considered suitable for cDNA conversion (Suppl. figure 2). Each RNA sample was converted to cDNA using the high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s specifications (Suppl. figure 3).

The primers for qPCR were designed using OligoAnalyzer software (Thermofisher). In total, eight primer pairs were designed for the experiment, three for endogenous genes and five for genes of interest (putative PAPs) (Suppl. Table 4). The primers were adapted to amplify PAPs from both Coffea arabica subgenomes by comparing homeolog sequences using Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Carabica_er), via Blastn tool and multiple alignments, using the ClustalW algorithm (Thompson et al. 1994).

The analysis of the amplification efficiency of the primers was performed using LinReg software (Ruijter et al. 2009; Tuomi et al. 2010) individually for each primer, in each sample of the experiment. For the specificity analysis of each primer pair, the behavior of the melting curve was analyzed in all samples, and only the primer pairs with single peaks were considered specific.

The analysis of gene expression via qPCR was performed on an ABI 7500 FAST equipment (Applied Biosystems), using the Gotaq qPCR Master Mix reagent (Promega). The stoichiometry and cycling specifications were adopted based on the manufacturer’s recommendations, the experiment was carried out in biological triplicates and the resulting data treated using the Pfaffl method (Pfaffl et al. 2004).

Statistical analyses

The data obtained in the experiment were subjected to analysis of variance (F test). The averages were compared using the Tukey test, at a significance level of 5%. The statistical software used were Genes (Cruz 2013) and R (R core Team 2020).

Results

A total of 29 genes putatively belonging to PAP family were identified on C. canephora genome, named from CcPAP1 to CcPAP29 accordingly to their genomic location (Table 1). From this set of genes, only two (CcPAP1 and CcPAP2) are located on the chromosome zero, which corresponds to the unmapped portion of the genome (Denoeud et al. 2014), and the chromosome 11 is the most enriched with identified PAP genes (about 38% of the total).

Table 1.

Description of the basic characteristic of putative PAP family members identified on C. canephora

Gene id Annotation Genome position Gene size Protein size Molecular weight
Cc00_g07780 CcPAP1 chr0:63352692..63358978 6287 bp 468 aa 53 kDa
Cc00_g33340 CcPAP2 chr0:194895365..194896699 1938 bp 271 aa 30 kDa
Cc02_g17140 CcPAP3 chr2:15786699..15789323 3148 bp 471 aa 54 kDa
Cc02_g29480 CcPAP4 chr2:30825788..30829969 4182 bp 267 aa 30 kDa
Cc02_g29510 CcPAP5 chr2:30881299..30892365 11,335 bp 645 aa 71 kDa
Cc03_g07240 CcPAP6 chr3:6502762..6505842 3805 bp 493 aa 55 kDa
Cc03_g07920 CcPAP7 chr3:7464215..7469516 5681 bp 542 aa 61 kDa
Cc04_g09270 CcPAP8 chr4:7728638..7730426 1995 bp 332 aa 38 kDa
Cc05_g13780 CcPAP9 chr5:27257151..27260337 3592 bp 440 aa 50 kDa
Cc06_g05610 CcPAP10 chr6:4506937..4509169 2300 bp 439 aa 50kDa
Cc06_g05620 CcPAP11 chr6:4515612..4518271 2969 bp 432 aa 48 kDa
Cc06_g07850 CcPAP12 chr6:6247774..6252934 6465 bp 478 aa 55 kDa
Cc06_g17480 CcPAP13 chr6:17468591..17474451 7192 bp 410 aa 46 kDa
Cc06_g18310 CcPAP14 chr6:18566604..18571791 5400 bp 392 aa 44 kDa
Cc06_g20820 CcPAP15 chr6:25707705..25710211 2632 bp 364 aa 42 kDa
Cc06_g20830 CcPAP16 chr6:25710298..25716098 5915 bp 265 aa 29 kDa
Cc08_g16870 CcPAP17 chr8:31282144..31287954 6351 bp 617 aa 69 kDa
Cc10_g02260 CcPAP18 chr10:1713861..1716980 4187 bp 426 aa 49 kDa
Cc11_g02390 CcPAP19 chr11:7595076..7599866 6212 bp 376 aa 42 kDa
Cc11_g05810 CcPAP20 chr11:21432170..21436831 4993 bp 331 aa 38 kDa
Cc11_g05820 CcPAP21 chr11:21442638..21449181 6835 bp 331 aa 38 kDa
Cc11_g05830 CcPAP22 chr11:21455190..21459018 3829 bp 387 aa 45 kDa
Cc11_g11960 CcPAP23 chr11:29152620..29157975 5356 bp 645 aa 73 kDa
Cc11_g12000 CcPAP24 chr11:29180929..29186028 5836 bp 611 aa 69 kDa
Cc11_g12010 CcPAP25 chr11:29187683..29193282 6130 bp 613 aa 69 kDa
Cc11_g12020 CcPAP26 chr11:29194409..29198730 5019 bp 611 aa 69 kDa
Cc11_g12240 CcPAP27 chr11:29363693..29366574 4099 bp 494 aa 55 kDa
Cc11_g12250 CcPAP28 chr11:29369432..29370043 853 bp 203 aa 23 kDa
Cc11_g12260 CcPAP29 chr11:29370109..29371299 1799 bp 231 aa 26 kDa
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The size of the putative translated proteins range from 203 to 645 amino acid residues (aa), but most of the members (62%) are on the interval of 300 to 550 a.a, and the theoretical pI varies from 4.63 to 8.84. The molecular weight of putative CcPAPs range from 23 to 73 kDa, and we have classified 12 of them as low molecular weight PAPs (less than 45 kDa) and the other 17 as high molecular weight PAPs (Tran et al. 2010a) (Table 1, Suppl. Table 1). The subcellular localization prediction for all the coffee PAP proteins was analyzed (Suppl. Table 1), and the most common prediction is “extracellular” (10 out of 29 proteins).

Although the number of exons and the annotated gene size vary greatly between the genes (Table 1, Fig. 1), all of the selected members have at least one characteristic conserved domain for PAP family (such as cl26054 and cl13995), observed on already characterized members on diverse plant species. The overall gene structure also varies between C. canephora putative PAPs (Fig. 1).

Fig. 1.

Fig. 1

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Gene structure of putative PAP members on C. canephora. Each exon is displayed as yellow boxes, introns as black lines and UTR regions as blue boxes

From this set of proteins, 15 have at least one of the five conserved motifs found on PAPs from other plant species, which are associated with the phosphatase function (DXG/GDXXY/GNH(D/E)/VXXH/GHXH; where underlined letters indicate metal-binding residues) and five putative C. canephora PAPs have all the conserved motifs with the exact displayed sequence, CcPAP1, CcPAP3, CcPAP11, CcPAP12 and CcPAP18 (Suppl. figure 4).

The protein sequences of the 29 putative CcPAPs were aligned with members of the same family on A. thaliana and O. sativa, and a phylogenetic tree was constructed where CcPAPs were distributed on three distinct groups (Fig. 2). Group 1 had seven C. canephora proteins allocated, group 2 had eleven and group 3, nine. Among CcPAPs, three sister groups were formed (CcPAP20/CcPAP21, CcPAP15/CcPAP17, CcPAP4/CcPAP5), other small cluster has CcPAP2, CcPAP27 and CcPAP29 and the biggest C. canephora PAP proteins cluster is composed by four members, CcPAP23, CcPAP24, CcPAP25 and CcPAP26. Overall, the PAP members from all the plant species were evenly distributed along the phylogenetic three, with no big clusters formed by proteins from the same species.

Fig 2.

Fig 2

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Phylogenetic three of PAP members from different plant species. “Cc” stands for C. canephora; “At” for A. thaliana and “Os” for O. sativa. C. canephora members are highlighted on red font

To elucidate whether some of the putative CcPAP genes could be associated with acid phosphatase activity on the rhizosphere of coffee plants, we have collected samples composed by several C. arabica genotypes submitted to contrasting P supply conditions. The range of phosphorus in the soil was 0.52–3.34 mg dm−3 without and with phosphate fertilization, respectively (Vilela et al. 2021). Firstly, the acid phosphatase activity was evaluated following Tabatabai and Bremner (1969) method (Table 2), and to distinguish the plant-derived phosphatase secretion from possible microbial-derived phosphatase, the microbial biomass carbon was also measured in each sample following the methodology proposed by Vance et al. (1987) (Table 3 supplementary).

Table 2.

Mean and standard deviation of acid phosphatase activity (FFT) of C. arabica genotypes rhizospheres in relation to phosphorus supply condition

Genotypes FFT (µg ρ-nitrophenol g−1 h−1)
− P + P
Bourbon Amarelo IAC J10 81.75 ± 24.93 ab A 54.39 ± 24.35 b A
Catiguá MG2 63.57 ± 40.02 ab A 83.31 ± 50.11 b A
Catuaí Vermelho IAC 144 85.34 ± 32.36 ab A 77.05 ± 18.36 b A
MGS Ametista 66.23 ± 51.30 ab B 190.77 ± 20.79 a A
MGS Aranãs 95.67 ± 22.27 ab A 57.47 ± 17.07 b A
MGS Paraíso 2 142.34 ± 29.25 a A 46.13 ± 31.43 b B
Paraíso MG H 419-1 123.29 ± 8.88 ab A 94.23 ± 33.94 b A
H 6-47-10 pl. 3 98.25 ± 62.14 ab A 120.52 ± 34.80 ab A
Sarchimor MG 8840 47.52 ± 29.91 b A 48.16 ± 20.57 b A
Topázio MG 1190 92.29 ± 36.16 ab A 98.43 ± 45.54 ab A
Mean 89.63 87.05
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Means followed by the same lower-case letter on the column and upper-case letter on the line do not differ statistically from each other, at 5% significance, by Tukey test

For acid phosphatase activity (FFT), there was a tendency for increase in this parameter in all treatments. However, opposite to what was observed with microbial biomass carbon (Table 3 supplementary), the acid phosphatase activity values were very similar inside the genotypes, with or without phosphate fertilization. The soil used in the experiment had an acid phosphatase activity of 68.55 µg ρ-nitrophenol g−1 h−1, with an average increase of 30.75% (89.63 µg ρ-nitrophenol g−1 h−1) without P fertilization and 26.99% (87.05 µg ρ-nitrophenol g−1 h−1) for fertilized rhizospheres. Lammel et al. (2015), working with different soil management systems in coffee trees, found values of acid phosphatase ranging from 4.6 to 8.3 mg ρ-nitrophenol g−1 h−1.

Analyzing the data presented in Table 2, it can be noted that the cultivar MGS Ametista had greater FFT under phosphate fertilization treatment, and in the cultivar MGS Paraíso 2, the behavior was the opposite. MGS Paraíso 2 had greater FFT in conditions of low P supply, indicating that the phosphatase activity in this cultivar could be activated upon P starvation stress. Still regarding FFT, Sarchimor MG 8840 had the lowest values for this characteristic and no variation among P supply conditions. Therefore, due to this contrast in APAse activity between MGS Paraíso 2 and Sarchimor MG8840 (Suppl. figure 5), these two cultivars were selected to investigate the expression of PAP genes, to determine whether selected members of this family could be associated with P starvation-induced phosphatase activity.

Five genes were selected as candidates for coding secreted purple acid phosphatases, which could be involved on the rhizosphere phosphatase activity, CcPAP1, CcPAP3, CcPAP11, CcPAP12 and CcPAP18, based on their localization on the phylogenetic tree (Group 3, Fig. 2), resembling their similarity with members of this family already characterized as rhizosphere-secreted acid phosphatases on other plant species (Tran et al. 2010b; Robinson et al. 2012; Tian et al. 2012; Lu et al. 2016; Mehra et al. 2017). These C. canephora sequences were compared to those from C. arabica genome, and the two putative homeologs (alleles from the different ancestral-derived subgenomes) found on this species were analyzed as one major representative gene for each C. canephora relative, named CaPAP1,3,11,12 and 18.

CaPAP1 was not expressed on the root tips from the two coffee cultivars, and for CaPAP3, CaPAP11 and CaPAP12, there was no statistically significant difference on gene expression among the P supply conditions within the cultivars tested. Only CaPAP18 had significant variation on gene expression, and it was 2.33-fold upregulated upon P starvation on the MGS Paraíso 2 cultivar (Fig. 3). This pattern of CaPAP18 gene expression variation coincides with the phenotypic differences observed—MGS Paraíso 2 with higher rhizosphere APAse activity on low P supply condition, although no increase in microbial biomass carbon was found and, the Sarchimor MG 8840 cultivar, with no increment in acid phosphatase activity.

Fig. 3.

Fig. 3

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Gene expression profile of selected PAP family members on two coffee cultivars submitted to different P supply conditions. “Paraiso-low P” stands for MGS Paraíso 2 cultivar on P starvation condition; “Paraíso” for MGS Paraíso 2 cultivar on normal P supply; “Sarchimor-low P” for Sarchimor MG 8840 cultivar on P starvation condition and “Sarchimor” for Sarchimor MG 8840 cultivar on normal P supply. Different letters on top of boxes indicate statistically significant variation between average values considering P-value < 0.05 and Tukey test

Discussion

To sustainably cope with future demands from agriculture, crops must be adapted to sustain high yields on low P fertilizer input conditions, as this is a non-renewable resource. Moreover, P efficient crops would absorb more Pi from the soil, avoiding pollution and extra costs; however, to this end, plants need to overcome the low mobility and availability of Pi. One of the mechanisms that compose the Pi starvation response and may be an important trait to explore as part of the strategy to develop P efficient cultivars is the secretion of PAPs on the rhizosphere (Gonçalves et al. 2020).

PAP family comprises enzymes that act on recycling internal reservoirs of P on the plant upon Pi starvation and some of them are secreted on the rhizosphere, helping on the solubilization of Pi, but they are also involved on other functions beyond those related to P starvation, such as salt tolerance responses (Wang and Liu 2018, Abbasi-Vineh et al. 2021), and we have identified 29 putative members of this family on C. canephora (Table 1). The number of PAP genes in C. canephora is close to other plant species, like A. thaliana (29, Li et al. 2002), O. sativa (26, Zhang et al. 2011) and G. max (35, Li et al. 2012). Xie and Shang (2018) analyzed the number of these genes on diverse vegetable species and found the following results for each of them: Brassica oleracea (44), Brassica rapa (37), Cucumis sativus (18), Cucumis melo (15), Citrullus lanatus (16), Solanum lycopersicum (27), Solanum pennellii (28), Solanum tuberosum (22), Solanum melongena (19) and Capsicum annuum (22). Despite the variation for some of these vegetable species, the close number of PAP genes on distantly related plant species such as O. sativa, A. thaliana and S. lycopersicum indicates that this family size does not fully correlate to plant lineage phylogenetic distance and their genome duplication events, and our result with C. canephora reinforces this observation.

This is also observed by analyzing the PAP phylogenetic tree (Fig. 1) presented here, as the members from different species are evenly distributed along diverse clusters, with low frequency of concise groups formed by PAPs within the same species, thus indicating low occurrence of lineage specific duplications. An exception of this pattern is observed for CcPAP23, CcPAP24, CcPAP25 and CcPAP26, as their translated proteins are similar, grouped together on the phylogenetic tree, the genes are close on the genomic context and their structure is similar (Fig. 1). Moreover, the last three mentioned members are side by side on the annotated genome, indicating a possible recent duplication which might be specific to C. canephora. Similar results were already found for other species (Srivastava et al. 2020; Gonçalves et al. 2020).

Three main groups are formed on the phylogenetic three of C. canephora and other plant PAPs. Group 3 is the biggest and contains most of the characterized PAPs that are secreted on the rhizosphere upon Pi starvation stress, like AtPAP12, AtPAP26, OsPAP10a, OsPAP10c and OsPAP21b (Tran et al. 2010b; Robinson et al. 2012; Tian et al. 2012; Lu et al. 2016; Mehra et al. 2017). Nine CcPAPs are located on this group, of which CcPAP1, CcPAP3, CcPAP12 and CcPAP18 are closely grouped with at least one of the above-mentioned characterized proteins (Fig. 2).

Due to the potential on coding secreted APAses, the genes that codify these four proteins, together with CcPAP11 (also located on group 1 and containing all the conserved motifs for PAP family), were selected as candidates for gene expression analysis on coffee plants submitted to variation on P supply conditions. The experiment was performed on a greenhouse, and the soil used had 0.28 mg dm−3 of available P (P-Mehlich-1 extractor), a common and low value of available P found on Brazilian Cerrado’s soils (Guimarães et al. 1999). The microbial biomass carbon value can be used as an indicator of sustainable management systems in coffee production. This is reinforced by the fact that among some biological attributes of the soil, Partelli et al. (2012) found in the microbial biomass carbon the variable with the greatest relative contribution to the discrimination between the conventional and organic production system in Coffea canephora.

In a study involving different cultivation systems, the lowest values of microbial biomass carbon were found in coffee plants grown in monoculture system, when compared with those intercropped with tree and grass species (Guimarães et al. 2017). In another study, Almeida et al. (2007), evaluating the influence of an agroforestry system on the microbial biomass of the soil, found that the highest levels of microbial biomass carbon were found in coffee growing areas in an agroforestry system and under native forest. Glaeser et al. (2010) evaluated the microbial biomass carbon in different coffee growing systems and at two times of the year and observed that the highest values were found in native vegetation, in dense coffee trees and in coffee plants intercropped with Musa spp. and Acacia sp.

The explanation for the greater acid phosphatase activity where there was phosphate fertilization was due to the improvement in the cultivation environment due to the addition of phosphorus, which is very important in the metabolism of all living beings, including soil microorganisms. A parameter that reinforces this statement was that the microbial biomass carbon showed the same behavior in the cultivar MGS Ametista (Table 2), showing that the improvement in the environment provided an increase in the microbial activity of the soil, the major responsible to produce soils acid phosphatase.

According to Dick (1994), the enzymatic activity of a soil is the result of the sum of the enzymatic activity of living organisms (microorganisms, animals and plants) and of enzymes associated with the nonliving fraction (abiotic enzymes that accumulate in the soil protected from proteases through adsorption on clay particles and organic matter). In most of the studied fungi, acid phosphatases are subject to repression by high concentrations of inorganic phosphate (Aleksieva and Micheva-Viteva 2000; Bernard et al. 2002). The secretion of extracellular enzymes is directly correlated with the extension and growth of hyphae (Hidayat et al. 2006). Enzymatic activity can be affected by soil management practices, with reductions occurring in conventional management systems, when compared to conservation management (Aon et al. 2001). This explains the fact that areas of native forest or more preserved have higher concentrations of phosphatase (Carneiro et al. 2008; Jakelaitis et al. 2008).

According to Dick and Tabatabai (1993), microorganisms would be the most expressive sources of phosphatases in the soil, because of their large biomass, high metabolic activity and short life span, with several generations, thus allowing the production and release high amounts of extracellular enzymes when compared to plants. In a study performed by Fernandes et al. (2000), the authors also found greater acid phosphatase activity in two types of soil with increasing doses of P. The lower activity observed in the lower doses of P applied is probably due to changes in the normal metabolism of plants, resulting from phosphate nutrition.

In a large study with compiled data on phosphatase research, Margalef et al. (2017) found a correlation between microbial carbon and organic P. Moreover, the authors found a relationship between the increase in microbial carbon and the increase in acid phosphatase, which means that the increase in acid phosphatase activity is closely linked to the increase in microbial biomass.

As for the cultivar MGS Paraíso 2, the explanation for the greater acid phosphatase activity when there was no phosphate fertilization is due to the ability of the cultivar itself to produce and exude acid phosphatase in its rhizosphere. In the case of the cultivar MGS Paraíso 2, there was a significant increase in the microbial biomass carbon when the supply of P via fertilization was carried out, the opposite result to that observed in relation to acid phosphatase activity, which was significantly higher when there was no supply of P via fertilization, showing that the observed acid phosphatase activity was due to the activity of the plant itself and not due to the soil microbiota. This observed result reinforces the data on the behavior of the gene expression of some PAP´s presented in Fig. 2, showing the fact that the cultivar MGS Paraíso 2, in P deprivation, is more efficient in the production of acid phosphatase.

Phosphatases can be constitutive or repressible by phosphate. Constitutive phosphatases are synthesized regardless of the composition of the medium in which they act; repressible ones are synthesized only in the presence of limiting concentrations of phosphate (Esposito and Azevedo 2004). As the supply of P for the plants decreases, there is an increase in the activity of these enzymes (Bieleski and Fergunson 1983). Some studies have reported the relationship between P deficiency and phosphatase activity (Ascencio 1994; Fernandes et al. 1998; Fernandez and Ascencio 1994; Silva and Basso 1993). According to Rössner et al. (1996), acid phosphatases are excreted by both plant roots and soil microorganisms. These enzymes are inducible and, therefore, are synthesized predominantly under conditions of low phosphorus availability in the soil.

In general, for the evaluated genotypes, the microbial biomass carbon was favored by the supply of phosphorus, showing significant increases in a short period of evaluation. The acid phosphatase release occurred dynamically, with differences in the behavior of the genotypes accordingly to phosphate fertilizer input variation. This result shows that the genetic variability within the coffee genotypes is relevant in the study of acid phosphatases, since there was an increase in the activity of this enzyme both in the environment that was fertilized with phosphorus and in the environment where the fertilization was not carried out.

From the five selected coffee PAPs, the CcPAP1 was found to not be expressed on the roots of both MGS Paraíso 2 and Sarchimor MG 8840 at normal and stressed conditions. Despite its C. canephora representing member, CcPAP1, codes for a protein that is located on the same phylogenetic group as the other tested PAPs (Group 3, Fig. 2), the closest characterized protein near it is AtPAP25, which is probably involved on Pi starvation-induced signaling pathway to respond the scarcity stress. Although there is similarity on the protein level with AtPAP25, CaPAP1 seems to not play a direct role on the root response to Pi starvation, at least not on the conditions we have analyzed. This gene can still be related to Pi starvation response by being involved on P recycling on other plant tissues, which requires further investigations. Still, members of this family are found related to other stimuli, like nutrient homeostasis during germination and flowering (Suen et al. 2015; Zhu et al. 2005).

The other four selected genes were expressed on coffee roots, although for three of them (CaPAP3, CaPAP11, CaPAP12), there was no significant differential mRNA accumulation between the conditions and cultivars tested here (Fig. 3). This observation does not exclude these PAP members of being involved on Pi scavenging process on the rhizosphere. Although our data show that they are not regulated upon Pi supply variation, they could still code for secreted phosphatases that are related to Pi uptake at non-specific stimuli or, otherwise be regulated on a different stress level than the one used on this work.

CaPAP18 was the only gene which its mRNA accumulation varied among the tested treatments on this work (Fig. 3). The 2.3-fold upregulation upon Pi starvation only on MGS Paraíso 2 cultivar is closely linked to the variation in phosphatase activity found on the soil for this cultivar and, interestingly, the C. canephora representing member CcPAP18 is the most similar one to AtPAP12, a known A. thaliana secreted purple acid phosphatase (Tran et al. 2010b; Wang et al. 2014). On A. thaliana, it was already reported a reduction in secreted APAse activity (culture medium) of about 40% upon Pi starvation conditions on plants which the AtPAP12 gene is mutated (pap12) and 15% less root-associated APAse activity on Pi starved plants, demonstrating that this gene is determinant for A. thaliana Pi starvation response, together with AtPAP26 and AtPAP10 (Wang et al. 2014). Considering that there was no observed increase in microorganism’s biomass on MGS Paraiso 2 rhizosphere on low Pi condition, it is possible to hypothesize that the increase in APAse activity might be derived from plant secretion of enzymes and a good candidate gene determining this characteristic would be CaPAP18.

The APAse activity directly impacts on plant development upon Pi starvation stress, as one of the main plant’s response to this stress. Arabidopsis thaliana pap12 plants had 10% less biomass on Pi-deprived conditions in comparison with WT ones, and events overexpressing AtPAP12 had increased biomass gain of 25–40% relatively to WT on the same P supply condition (Wang et al. 2014). Conversely, when overexpressing one ortholog of a main regulator of Pi starvation response in apple, MdPHR1, transgenic plants had higher APAse activity and higher Pi content upon Pi-deprived conditions, possibly due to higher transcription of MdPAP10 (Li et al. 2020).

This effect on plant development can be followed by increase in yield, as reported for rice events overexpressing OsPAP10c under the control of its native promoter, where transgenic plants had greater vegetative biomass (both root and shoot) accumulation in comparison with WT ones, but also higher values of grain yield per plant (Deng et al. 2020). Therefore, beyond the possibility of developing a molecular marker for coffee tolerance to low Pi conditions, the analysis presented on this work, especially for CaPAP18, might pave the way for deeper understanding of PAP genes influence on Pi uptake for the coffee plant and integrate future biotechnological strategies to improve the adaptability of this crop to low Pi input cultivation environment.

Much of the area used for coffee cultivation in Brazil is characterized by acidic soils with low natural fertility, where successive fertilizer inputs are required to obtain high yields. The cultivar MGS Paraíso 2, being efficient on the use of phosphorus (Vilela et al. 2021), in part, probably due to this new observed characteristic (greater production of acid phosphatase), can be used in low phosphate input plantations, and the gene explored in this work might compose an important biotechnological strategy to produce coffee sustainably.

Conclusions

Twenty-nine genes from the PAP family were identified in the Coffea canephora genome.

In the cultivar MGS Paraíso 2, some PAPs were more expressed under P deprivation (CaPAP3, CaPAP11 and CaPAP18) and others were indifferent to the question of P (CaPAP12).

CaPAP18 gene expression and acid phosphatase activity were highly significant in the cultivar MGS Paraíso 2 under P deprivation.

Phosphate fertilization positively influences the microbial biomass carbon.

Arabica coffee genotypes respond positively both with phosphate fertilization (MGS Ametista) and without phosphate fertilization (MGS Paraíso 2) in acid phosphatase activity.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (JPG 52 KB) (52KB, jpg)
Supplementary file2 (JPG 81 KB) (80.5KB, jpg)
Supplementary file3 (JPG 43 KB) (42.9KB, jpg)
Supplementary file4 (JPG 5399 KB) (5.3MB, jpg)
Supplementary file5 (JPG 59 KB) (59KB, jpg)
Supplementary file6 (XLSX 16 KB) (16.4KB, xlsx)
Supplementary file7 (DOCX 25 KB) (25.4KB, docx)
Supplementary file8 (DOCX 25 KB) (24.6KB, docx)
Supplementary file9 (XLSX 11 KB) (11.1KB, xlsx)

Acknowledgments

To FAPEMIG, CNPq and Consórcio Pesquisa Café (Embrapa/Café) for financial support. To CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for granting the scholarship. “This work was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Financing Code 001”.

Author contributions

DV, RP and TC carried out the experiments, analyzed the data and wrote the manuscript. LP, MAC and GC designed the research and edited the manuscript. JS designed the research, carried out the experiments and analyzed the data.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Contributor Information

Diego Júnior Martins Vilela, Email: diegovilela26@yahoo.com.br, Email: diego.vilela@epamig.br.

Renan Terassi Pinto, Email: renantpinto@gmail.com.

Thiago Bérgamo Cardoso, Email: thiagobergamoc@gmail.com.

Luciano Vilela Paiva, Email: luciano@ufla.br.

Marco Aurélio Carbone Carneiro, Email: marcocarbone@ufla.br.

Gladyston Rodrigues Carvalho, Email: grodriguescarvalho@gmail.com.

Jessé Valentim dos Santos, Email: jessevalentim@gmail.com.

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Supplementary Materials

Supplementary file1 (JPG 52 KB) (52KB, jpg)
Supplementary file2 (JPG 81 KB) (80.5KB, jpg)
Supplementary file3 (JPG 43 KB) (42.9KB, jpg)
Supplementary file4 (JPG 5399 KB) (5.3MB, jpg)
Supplementary file5 (JPG 59 KB) (59KB, jpg)
Supplementary file6 (XLSX 16 KB) (16.4KB, xlsx)
Supplementary file7 (DOCX 25 KB) (25.4KB, docx)
Supplementary file8 (DOCX 25 KB) (24.6KB, docx)
Supplementary file9 (XLSX 11 KB) (11.1KB, xlsx)

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