Proteolysis of seed storage proteins (SSP) during germination provides a steady supply of amino acids to the embryo development into seedling. This process is coordinated by different peptidases that act sequentially and overlaid mode. These enzymes are an ancient group evolved separately in a wide structural and functional diversity and have many applications in medicine, pharmacy and industry. However, the knowledge about seed peptidases during germination w as obtained from studies almost restricted to the cultivated species. This restriction implies caution about generalizations made from these studies, as well limit s the biological knowledge about plant kingdom and technological use from plant peptidases. In this work, a scan of the proteolytic activity was held in germinating seeds of a leguminous subtropical woody tree. Eleven proteolytic activities were detected in protein extracts from embryonic axis and cotyledons. The presence and intensity of these activities varied over time and between these tissues. There was indication that aspartyl-endopeptidases (phytepsins) and cysteine-carboxypeptidases (plant cathepsins) were involved in A. colubrina SSP hydrolysis. These peptidases differ to that commonly involved in germination of the cultivated leguminous. In addition, one of detected phytepsins showed stability on pH scale, which is important for industrial uses. There was also detected a metallo-carboxypeptidase activity, which has been not described in plants. These peptidases must be isolated to confirm or not these indications. However, these data indicate the biological and technological importance of extending the studies about plant peptidases on a diverse genetic basis.
The proteolysis of the seed storage proteins (SSP) during germination is crucial to seedling establishment. This process, which is activated at seed hydration and remains active after germination, is coordinated by joint activity of different peptidases. These hydrolases are expressed in temporal- and tissue-specific way. They are accumulated in embryonic axis and reserve tissues during seed maturation, as well synthesized during and after germination. Thus, being activated or synthesized, these enzymes sequentially hydrolyze SSP and provide continuous amino acids supply for resumption of embryonic axis development until seedling establishment [
The explanatory models of this proteolytic process were based from studies with some cultivated species. Despite the validity of these models, the restriction of studies to these few and genetically standardized plants limits the biological knowledge about proteolysis during germination and other plant proteolytic processes. Maybe is enough to consider the close genetic base that the model plants represent in front of the angiosperms diversity [250,000 species in the world; 50,000 in Brazil] [
Peptidases originate from 60 evolutionary lines that resulted in an extensive multifunctional group of enzymes [
Indeed, at germination, these enzymes vary even in model plants. The hydrolysis routes of SSP among different plants show more similarities to the SSP cleavage sites than the peptidases that cleave then [
Plant peptidases show direct involvement in essential cell cycles [
On the other side, from works with peptidases active in germination, the majority focuses isolated enzymes. In spite of the obligatory use of purified enzymes to determine their structures, specific functions, evolutionary relations and technological applications, works that exploit germination in relation to whole proteolytic activity are rare. But it is important to expand the plant peptidases research on a diverse genetic basis to increase knowledge about proteolysis in germination, as well in other plant proteolytic processes; and to explore the technological use that plant peptidases may have [
In this way, our objective was to carry out a scan of the proteolyticactivity during and after germination inseeds of a subtropical woody species native to South America. Woody species represent the ecological climax of most terrestrial ecosystems, but there is little biochemistry information about its germination, especially from the tropical and subtropical ones. However, A. colubrina (Fabaceae), called Angico in Brazil, has pharmacological [
In this context, we used synthetic peptide substrates and peptidase inhibitors to detect, quantify and partially characterize, via spectrofluorometry and high performance liquid chromatography, the activity of peptidases in protein extracts from A. colubrina embryonic axis and cotyledons separated before, during and after germination.
A. colubrina seeds were supplied by LAS/UFLA, from a batch of seeds randomly collected from several local trees (21˚14'42''S 45˚00'00''W). Visibly normal and undamaged seeds were placed to germinate on rolls made from 3 sheets of germitest paper soaked in distilled water at volume 2.5 times the dry paper weight. These seed rolls were kept at 30˚C, without photoperiod control, during 0, 6, 12, 18 and 24 hours (h). Fifty seeds were used for each hydration time. Seeds apparently dead or presenting necroses were not collected. The seed coats from the remaining seeds were discarded, embryonic axes (E) and cotyledons (C) were separated and collected for immediate protein extraction. Collected tissues from E and C were macerated using liquid N2. The powder was homogenized with ice-cold Tris buffer [50 mM/pH 7.5] at 25 mg E or 100 mg C to 1 mL buffer. The homogenate was maintained at 4˚C for 2 h under occasional shaking and then centrifuged twice at 16,000 g for 20 min at 4˚C. The supernatant containing water-soluble proteins (crude extract) was diluted 1:1 with glycerol (v/v) and stored at −40˚C. The protein content of these extracts was estimated by Bradford method [
The proteolytic activity (At) in E and C samples was detected by spectrofluorometry using FRET (fluorescence resonance energy transfer) synthetic peptide substrates that emit fluorescence when hydrolyzed1. The substrates used were Abz-AIAFFSRQ-EDDnp, Abz-FRAK(Dnp)-OH, F-MCA and Z-FR-MCA. These substrates were synthesized according INFAR protocol [
A t = V h K n ⋅ p t × 1000
where At is proteolytic activity (nM∙μg∙min−1), Vh is hydrolysis rate (AFU/min), Kn is K constant for each fluorescent group (AFU/μM) and pt is the amount of protein in the sample (μg)2. All reactions were performed in duplicate.
1By resonance to Z, EDDnP and Dnp quenching groups, Abz and MCA fluorescent groups exhibit low fluorescence that increases when the substrate molecule is ruptured.
2The spectrofluorometer was adjusted to record the fluorescence every 0.4 s during 10 min. With increasing amount of fluorescence over time, the equipment generates an accurate linear regression (R2 > 0.99), whose angular coefficient corresponds to hydrolysis speed and represents a direct measure of the proteolytic activity.
After obtaining At data, were chosen 6 results with Abz-AIAFFSRQ-EDDnp, 3 with Abz-FRAK(Dnp)-OH, 8 with F-MCA and 3 with Z-FR-MCA to repeat the reactions that gave rise to these 20 results, but now using peptidase inhibitors. These results were chosen due At expressivity from individual samples and/or particular variations of At in time profile and/or pH profile. The peptidase inhibitors used were 1 μM Pepstatin, 5 μM E-64, 500 μM PMSF or 5 mMo-phe, which are aspartyl-, cysteine-, serine- and metallo-peptidases inhibitors, respectively. The reactions were performed with protein solutions (4 μg∙mL−1) been incubated in absence or presence of inhibitors for 10 min at 37˚C under stirring. Thereafter, substrate was added and its hydrolysis was monitored for 10 min. The results were determined by Equation (1) and converted at percentage of inhibition relative to the control. The inhibitionhydrolysis of Abz-FRAK(Dnp)-OH by o-phe was performed by high performance liquid chromatography (HPLC).
To quantify the inhibition of Abz-FRAK(Dnp)-OH hydrolysis by o-phe, proteins solutions (8 μg∙mL−1) containing or not 5 mM o-phe were incubated at 37˚C for 10 min under stirring. Thereafter, 30 μM of substrate was added and aliquots (240 μL) were collected at 0, 7.5, 15, 30 and 60 min (serial hydrolysis). At each collect, the reaction was immediately stopped with 10 μL of trifluoroacetic acid (TFA) 1 M and stored at −40˚C. From these aliquots, 150 μL were injected into Shimadzu chromatograph with SPD-10AV UV-Vis and RF-535 fluorescence detectors coupled to Ultrasphere C-18 column (5 μm/4.6 mm × 150 mm) equilibrated with solvent [90% acetonitrile + 0.1% TFA (v/v)] at 10%. Samples were solvent-eluted in 10% - 80% gradient at 1 mL∙min−1 for 28 min and the column eluates were monitoredby their absorbance at 220 nm and by their fluorescence emissionat 420 nm following excitation at 320 nm. As chromatographic standards, were injected 150 μL of: 10 μM hydrolyzed substrate by sample for 15 h at 37˚C; 10 μM non-hydrolyzed substrate; 8 μg∙mL−1 protein + 40 mM TFA; and 8 μg∙mL−1 proteins + 40 mM TFA + 5 mM o-phe. Linear regression curves were constructed with the data of product amount from serial hydrolysis and the results were expressed as percentage inhibition relative to control without inhibitor.
The proteolitic activity (At) is a peptidic substrate function and there is no relation between quantification data and peptidic substrate. Thus, all of the at values from the same substrate were divided by the sample value that displayed maximum At and were rented as Relative Activity (Ar = At/Atmax). Peptidases class was chosen according to the inhibition percentage that each peptidases inhibitor had on tested At. Therefore, the data were also relatively expressed. After the individual analyzes with each substrate we obtained a tissue-temporal map of the proteolytic activity in A. colubrina seeds.
In this work, we perform an exploratory mapping of the peptidase activity in A. colubrina during and after germination seeds. The experiments were performed with crude protein extracts and synthetic substrates not with isolated peptidases and purified seed storage proteins (SSP), which would allow discerning, qualifying and quantifying the activity of specific peptidases. Therefore, no clear distinction was made among peptidases with specific function in SSP hydrolysis from peptidases related to other cellular processes, in spite of the activities were detected at germination. For the same reason, no distinction was made between possible isoforms. Although many peptidases are common to embryonic axis(E) and cotyledons (C), since cotyledonary tissue is embryonic tissue, there are isoenzyme members differentially present between these tissues [
In addition, radicle protrusion was used as criterion for germination of the seed batch. By this criterion, 4% of the seeds germinated at 12 h, 58% at 18 h and 93% at 24 h by hydration time (data not shown). No seed germinated in 6 h. Thus, the period up to 12 h refers to the beginning of the seed batch germination, 18 h at its end (>50%) and 24 h to post-germination events. On the other side, the proteolytic activity detected at 6 h was intermediate between 0 and 12 h and that detected at 18 h were intermediate between 12 and 24 h, both for E and C samples. Thus, the proteolytic data of 6 h and 18 h were not shown, being sufficient for analysis the times 0, 12 and 24 h as representative of the proteolytic events occurred respectively before, during and after germination of A. colubrina seeds.
Using Abz-AIAFFSRQ-EDDnp, which is specific for detecting endopeptidases, maximal activities were verified at pH 3 (
samples | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) |
---|---|---|---|---|
Pepstatin/Aspartyl | E-64/Cysteine | PMSF/Serine | o-phe/Metallo | |
E0 | 100 (3) | null (5; 7) | - | - |
E12 | 100 (3; 4) | - | - | - |
E24 | 100 (3) | - | - | - |
C0 | 100 (3) | - | - | - |
C24 | 100 (3) | null (5; 7) | - | - |
Plant aspartyl-peptidases (AP), or phytepsins, are endopeptidases specifically inhibited by pepstatin. They have maximum activity at acidic pH on several organic or synthetic substrates and show preference to hydrolyze peptide bonds formed by hydrophobic residues [
The temporal activity profile versus pH activity profile in Abz-AIAFFSRQ-EDDnp hydrolysis suggests the presence of three APs differentially expressed by the tissues (
These results suggest a sequential events where AP1 initiates the proteolysis in the protein bodies in embryonic axis and cotyledons, which is followed by AP2 mRNAs translation and after by AP3 mRNAs transcription and translation, mainly in cotyledons. The indication of these three APs in A. colubrina and the events sequence suggested is in accordance with the general seed pattern to store inactive forms of peptidases and their respective mRNAs during its maturation, as well to transcribe new mRNAs after germination. These suggest that peptidases and mRNAs stored in the embryos are sufficient for germination and that the largest mass of SSP is hydrolyzed from the storage tissues at post-germination events [
In fact, APs are accumulated in protein bodies [
The hydrolysis of Abz-FRAK(Dnp)-OH, which was designed for detect carboxypeptidase activities, shown that the activity increased with time in both tissues, but in different pH profiles (
The indication of AP and CP presences were respectively based on the relative spectrofluorometric quantification of Abz released during Abz-FRAK(Dnp)-OH hydrolysis from samples pre-incubated with pepstatin or E-64 (
Samples | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) |
---|---|---|---|---|
Pepstatin/Aspartyl | E-64/Cysteine | PMSF/Serine | o-phe/Metallo | |
E24 | 36 (4) | 57 (4); 3nr (7) | 1nr (7) | HPLC (4; 7) |
C24 | 14nr (4) | 87 (4) | - | HPLC (4) |
ht | E24/pH4 | C24/pH4 | E24/pH7 | ||||
---|---|---|---|---|---|---|---|
i | c | i | c | i | c | ||
Rt 11.8 | 7.5 | 100,188 | 274,256 | 568,197 | 964,183 | 166,120 | 407,029 |
15.0 | 126,810 | 326,520 | 951,756 | 1,467,040 | 325,765 | 556,018 | |
30.0 | 239,538 | 528,838 | 1,075,611 | 1,969,896 | 530,966 | 852,088 | |
60.0 | 387,110 | 905,879 | 1,136,122 | 2,389,160 | 993,327 | 1,485,255 | |
α/R2 | 5601/0.991 | 12,320/0.996 | 8669/0.623 | 25,171/0.896 | 15,432/0.997 | 20,559/0.999 | |
Rt 12.3 | 7.5 | 223,359 | 256,982 | 201,983 | 272,680 | 39,393 | 62,147 |
15.0 | 316,272 | 325,827 | 423,603 | 388,173 | 0 | 63,848 | |
30.0 | 536,795 | 519,355 | 451,907 | 503,666 | 0 | 76,122 | |
60.0 | 925,441 | 911,347 | 644,766 | 659,826 | 0 | 99,579 | |
α/R2 | 13,447/0.999 | 12,643/0.998 | 7218/0.854 | 6961/0.955 | 740/0.990 | ||
Rt 12.7 | 7.5 | 0 | 51,812 | ||||
15.0 | 0 | 68,870 | |||||
30.0 | 0 | 114,616 | |||||
60.0 | 0 | 200,842 | |||||
α/R2 | 2872/0.999 | ||||||
Qt | 2,855,513 | 4,049,004 | 5,453,945 | 8,614,624 | 2,055,571 | 4,038,226 | |
Qr | 0.33 | 0.47 | 0.63 | 1 | 0.24 | 0.47 | |
Inhibition (%) | 29 | 37 | 49 |
quantification of three fragments released from this hydrolyzed substrate by samples pre-incubated with o-phe (
With hydrolysis inhibition of Abz-FRAK(Dnp)-OH was observed carboxypeptidase activity of APs, CPs and MPs at acidic and neutral pH. The detected acidic CPs activity could be related with plant cathepsin-like. Although cathepsins are usually endo-CPs from papain family, several human cathepsins have preference to Lys (K) and Arg (R), as indicated by assays with a large substrates set [
acid (ABA) and gibberellin (GA) in aleurone layers and its expression increases with germination, as well in Arabidopsis, were a gene for cathepsin B-like, AtCathB3, is expressed at germination and post-germination events in embryonic axis and cotyledons, respectively [
According to MEROPS database [
MEROPS database [
By the data set from Abz-FRAK(Dnp)-OH hydrolysis, it seems more plausible to suppose that, at acid pH, o-phe inhibited not specifically the activity of some carboxy-CP (possibly a cathepsin-like) and that, at neutral pH, specifically inhibited the activity of some carboxy-MP, not yet described in plants.
The substrate F-MCA is generally used to detect aminopeptidases and the results shown that the major activities occurred at pH 7 (
The temporal activity profile in E samples describes an increase in activity during germination, followed by decay after that, mainly at pH 7. By the other side, the temporal activity of C samples profile was constant (
Plant metallo-aminopeptidases (amino-MPs) are distributed into M1, M17 and M24 families [
Samples | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) |
---|---|---|---|---|
Pepstatin/Aspartyl | E-64/Cysteine | PMSF/Serine | o-phe/Metallo | |
E0 | - | - | 38 (7) | 76 (7) |
E12 | - | - | 35 (7) | 77 (7) |
E24 | - | 19nr (7); 5nr (8) | 47 (7); 13nr (8) | 74 (7); 73 (8) |
C0 | - | - | 32 (7) | 77 (7) |
C12 | - | - | 32 (7) | 80 (7) |
C24 | - | 0 (7); 13nr (8) | 39 (7); 14nr (8) | 77 (7); 75 (8) |
hydrolyze F-MCA at neutral pH are: leucilaminopeptidase (M17), which is related to wounds and pathogens responses and it was detected in barley [
The hydrolysis of Z-FR-MCA, generally substrate for CP and SP, was more expressive in the E samples, in which the activity increased with time and pH scale (
indicating presence of MP, whereas in E24, it was also inhibited by E-64 and PMSF, indicating presence of CP and SP, respectively (
Legumains are a group of endo-CP related to storage and hydrolysis of globulins in some model plants. One of the major evidences for this is the fact that they are stored together with globulins in the acidic environment of the protein bodies [
Due to the low proteolytic activities in C samples (
Samples | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) | Inhibitor/Peptidase (class) |
---|---|---|---|---|
Pepstatin/Aspartyl | E-64/Cysteine | PMSF/Serine | o-phe/Metallo | |
E0 | - | 23nr (8) | 8nr (8) | 52 (8) |
E12 | - | 21nr (8) | 14nr (8) | 56 (8) |
E24 | - | 52 (8) | 56 (8) | 52 (8) |
A. colubrina cotyledons. Another possibility to explain this tissue-temporal profile of neutral-alkaline MP in A. colubrina takes place by the action of plant matrix metallo-peptidases (MMP). MMP are cytoplasmic endo-MP associated with cell membrane remodeling in developmental and/or wound processes [
Among plant SP, a reasonable assumption is the trypsin-like plant oligopeptidase-B (SC clan, S9 family, MEROPS classification). This enzyme is expressed in quiescent and germinating embryos of wheat and cleaved Z-FR-MCA with maximum activity at pH 8.5 [
Using fluorescent peptides and peptidase inhibitors, we detected several proteolytic activities associated with aspartyl-, cysteine-, metallo- and serine-peptidases (AP, CP, MP and SP, respectively) in protein extracts from A. colubrina embryonic axis (E) and cotyledons (C) excised before, during and after germination. The experimental conditions do not allow to directly relating the observed activities in E and C samples with specific germination events, since they do not allow discerning isolated peptidases, specific functions or native substrates. However, it was possible to make inferences about the possible peptidase identity related to the activities detected. These inferences were made from the determination of the catalytic class (based on pH of enzymatic activity, substrates used and peptidase inhibitors effectiveness) and from the tissue-temporal profile of seed proteolytic activity.
Considering experimental conditions, we speculate that phytepsins (AP group) initiate the SSP hydrolysis in A. colubrina. In cultivated dicots, the most common enzymes in SSP hydrolysis are CPs. Legumains and other CPs are involved in reserve globulin processing during seed maturation and its hydrolysis within the acidic protein bodies during germination [
Although non-detection of CPsusing these substrates does not exclude the possibility of CPs presence in A. colubrina seeds, phytepsins have been directly associated with SSP hydrolysis, by their presence in protein bodies [
3P1 refers to the 1st residue of sessile bond in the substrate accommodated by the 1st residue of enzyme catalytic site at N-terminal orientation.
However, Shutov and Vaintraub [
In the case of carboxy-MP activity detected by the hydrolysis of Abz-FRAK(Dnp)-OH, it was not possible to suppose some specific enzyme, since carboxy-MP is not described in plants, although gene sequences for several plant species are deposited in MEROPS databank. Carboxypeptidases that act with endo-CP in the globulin hydrolysis are SP (carboxy-SP) [
In general, these information agrees with the observations of Tan-Wilson and Wilson [
On the other hand, none of neutral-alkaline peptidases detected appear to be involved in cytoplasmic phase of SSP final degradation in A. colubrina. Cytoplasmic amino- and di-peptidases from metallo and serine classes are involved in final degradation of the oligopeptides generated by acidic endo- and carboxy-peptidases activities within protein bodies [
We generated initial information about the proteolytic activity in A. colubrina germinating seeds. Our results showed that A. colubrina germination presents different peptidases from that commonly found in cultivated leguminous. In addition, we possibly detected a peptidase with useful characteristics for industrial purposes and another one not yet described in plants. This study reinforces the need to amplify the detection of plant peptidases on a diverse genetic basis, since the research focused on commercial species is a limiting factor for the knowledge of proteolysis in germination, as in other proteolytic processes in the plant kingdom, and the potential technological use of plant peptidases. In this context, the scanning method chosen for this study can be applied in comparative studies of germination between different species and in other biological processes aiming wide detection of peptidases.
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes), Universidade Federal de Lavras (UFLA), Instituto Nacional de Farmacologia (INFAR), Universidade Federal de São Paulo (UNIFESP).
The authors declare that they have no conflict of interest.
Barduche, D., do Livramento, K.G., Judice, W.A.S., Paiva, L.V., Neto, L.J. and Guimarães, R.M. (2018) Proteolysis in the Subtropical Woody Tree Anadenanthera colubrina (Angico) Seeds during and after Germination. American Journal of Plant Sciences, 9, 1169-1190. https://doi.org/10.4236/ajps.2018.96088