Carbohydrate metabolism is critical for male fertility. Carbohydrate reserves (soluble sugars and starch) were quantified in three advanced stages of pollen development, from the final maturation to rehydration, in a chili pepper cultivar (Capsicum annuum L. cv. Calypso Red) with partially dehydrated pollen, to define possible carbohydrate inter-conversions. At the same time, the activity of soluble enzymes involved in sucrolysis and amylolysis were quantified to reveal potential metabolic pathways. The carbohydrates found (sucrose, glucose, fructose, and starch) apparently inter-convert. There would be a close relation between enzymatic activities and substrates, and the carbohydrate reserves could be key factors on the regulation of enzymatic activities. All the enzymes tested were active, although the relevance of each one changed along pollen maturation and rehydration, defining different profiles of enzymatic activities for each stage. Some usually neglected enzymes (e.g. soluble neutral invertase) have shown an important role in the stages analyzed, suggesting alternative processes to evaluate in the studies of male fertility control.
Functional pollen is needed to successfully complete fertilization in plants. Pollen functioning relies on several characteristics, which can differ among species. The amount of water and the carbohydrate reserves are biochemical features considered relevant for pollen viability and longevity [1,2]; both features would also be related to each other (e.g. high water content and scarce sucrose would determine a short living pollen [
An analysis of the content of carbohydrate reserves as well as a study of the activity of several soluble enzymes involved in their metabolization was carried out in different advanced pollen stages in a hot chili pepper (Capsicum annuum L.) with partially dehydrated pollen. The aim of this work was to analyze the possible inter-conversion between the carbohydrates studied and reveal potential metabolic pathways, in an attempt to contribute to the understanding of how carbohydrate utilization would be involved in the male functioning throughout the final steps of maturation up to rehydration.
Plants of the hot chili pepper (C. annuum) cv. Calypso Red were grown in a greenhouse at 32˚C ± 2˚C (optimal for flowering in this cultivar). Three developmental stages were studied: 1) immature bicellular pollen, collected one day before flower anthesis (1 DBA), 2) mature pollen collected the day of flower opening, and 3) rehydrated pollen, consisting of mature pollen rehydrated in vitro for 30 min, when pollen tubes started to emerge. The culture medium of Mercado et al. [
Pollen water content was calculated indirectly based on the difference in volume between fresh (for 1 DBA and mature pollen)/rehydrated and desiccated pollen (2 h at 100˚C); samples were submerged in immersion oil after being collected/treated. The formulas used to calculate the volume were:πr3 for spherical grains, andπ (longest r × shortest r2) for prolate spheroidal grains, where r is the radium. The average volume was calculated from 100 pollen grains in each stage. The difference between the volume of fresh/rehydrated and desiccated pollen would represent the volume filled up by water, i.e. the percentage of water. The latter values were used to calculate the dry matter content at each pollen stage and the final weight for the rehydrated pollen.
For carbohydrate analysis, the samples were homogenized in distilled water (1 DBA and mature stages) or in the culture medium (rehydrated pollen) with a PRO200 homogenizer, were inactivated by heating in boiling water for 10 min, and then centrifuged at 12,000 g and 4˚C for 25 min. The supernatants were used to quantify soluble carbohydrates while starch was quantified in the insoluble residues after three washes with distilled water. Samples were stored at –80˚C. Each stage was analyzed by triplicate. Soluble carbohydrates were identified and quantified through HPLC analysis, using single sugars as standards, according to Carrizo García et al. [
For the analysis of enzymatic activities, samples were prepared in an extraction buffer consisting of 1 mM EDTA, 1 mM DTT, 50 mM sodium phosphate, 3 mM MgCl2, and 2% glycerol (pH 7.4). The samples were homogenized and the supernatants were separated as described above. The activities assayed were soluble acid (Sai) and neutral (Sni) invertases, sucrose synthase in the cleavage direction (Susy), and amylases. Aliquots of every sample were incubated in a specific reaction medium for each enzyme. The reaction media were the following: 200 mM sodium acetate (pH 4.0) and 10 mM sucrose for the Sai; 50 mM sodium phosphate (pH 7.3) and 10 mM sucrose for the Sni; 50 mM MES (pH 6.5), 10 mM sucrose and 10 mM UDP for the Susy; 25 mM MOPS (pH 6) with solubilized starch (1 mg·mL–1 for 1 h at 100˚C) for the amylases. The mixtures were incubated 1 h at 30˚C in all the cases; the reactions were stopped by heating at 100˚C. Every enzymatic reaction was done three times for each stage. For the sucrolytic enzymes, sucrose hydrolysis was estimated through the changes of glucose and fructose. For the amylases, the quantity of starch hydrolyzed was calculated according to the changes of glucose and maltose. Glucose, fructose and maltose were identified and quantified as described above. Enzymatic activities were expressed as the amount of sugars released for min and total protein mg; total proteins were quantified according to Bradford [
Mean values were calculated for the substances quantified and the enzymatic activities assayed at all three stages. Data from consecutive stages were compared pair wise using ANOVA (P = 0.05).
The pollen water content fluctuated along stages in the chili pepper studied. The water content was ca. 45% at 1 DBA, when the pollen was spherical, and then diminished to 25% in the mature pollen, which acquired a prolate spheroidal shape (
from 1 DBA to the mature pollen, while glucose and fructose increased significantly, ca. 2.7 and 3-fold respectively (
The carbohydrate variations along the stages analyzed would evidence possible inter-conversions between the different substances quantified, considering that the pollen would be independent from the rest of the plant by 1 DBA [
In parallel to the carbohydrate temporal fluctuations, all the enzymatic activities quantified also changed. The sucrolytic activities increased from 1 DBA to the mature pollen, matching the reduction of the absolute sucrose concentration, and later decreased along with sucrose depletion during rehydration (
Amylolytic activities decreased significantly to one third from 1 DBA to the mature pollen, and then slightly, not significantly, during rehydration (
would decrease significantly between stages whereas the α-amylase activity would increase significantly (
It seems to be a logical inter-conversion between the carbohydrates analyzed. Besides, there would be a close relation between enzymatic activities and substrates, and the carbohydrate reserves (and perhaps exogenous nutrients when present) could be key factors on the regulation of enzymatic activities. All the enzymes tested were actively involved, in different and variable degrees, in pollen maturation and rehydration. Each stage was characterized by a different profile of enzymatic activities, in which the relevance of each enzyme was different. This trait could indicate different main pathways (and sites) of the carbohydrate reserve metabolization in each stage. The current results present alternative processes to evaluate, such as sucrolysis by the Sni and amylolysis by the β-amylase, in the studies of male fertility control.
CCG thanks the financial support of CONICET and FONCyT (Argentina). Italian authors are indebted to Piano di Ricerca dell’Ateneo (Siena University, Italia) for founding pollen research.
DBA—Day before Anthesis;
Sai—Soluble Acid Invertase;
Sni—Soluble Neutral Invertase;
Susy—Sucrose Synthase.