What if a plant tissue that lacks latex can still make a powerful antifungal mixture—but uses different molecular versions of the same defense proteins? That is the puzzle this study tests. The study asked whether callus cultures of Calotropis procera and Cryptostegia grandiflora reproduce the antifungal defense system typically found in latex.

Protein fractions from latex and callus tissues were evaluated against four fungi, while the presence of pathogenesis-related proteins was investigated. Calotropis procera callus proteins inhibited mycelial growth and spore germination of all tested fungi, but Cryptostegia grandiflora callus proteins showed no detectable antifungal activity.

The abstract links the activity to peptidases and osmotins, not peptidase inhibitors and chitinases, and indicates that callus and latex contain distinct protein isoforms. Laticifers are specialized secretory structures that produce latex, a complex mixture of metabolites and proteins rapidly released after tissue damage.

Latex is widely distributed among vascular plants and occurs in approximately six percent of plant species, especially in families such as Apocynaceae and Euphorbiaceae. Several pathogenesis-related protein classes have been reported in latex, including peptidases, chitinases, beta-one, three-glucanases, osmotins, and peptidase inhibitors.

These proteins contribute to plant defense through mechanisms including fungal cell-wall degradation, plasma-membrane disruption, inhibition of herbivore digestive enzymes, and induction of oxidative stress in pathogens. Cysteine peptidases are among the most abundant enzymatic classes in latex and have been widely associated with antifungal activity.

Osmotins, members of the PR-five protein family, inhibit fungal growth through membrane permeabilization, oxidative stress, and induction of programmed cell death. Despite extensive characterization of latex defense proteins, it remains unclear whether their genes are expressed beyond laticiferous tissues and whether the corresponding protein forms are conserved across tissues.

Callus provides a useful model because it is an undifferentiated tissue that lacks specialized secretory structures such as laticifers. If latex proteins are constitutively expressed, they should also appear in callus; if biosynthesis depends on laticifer differentiation, they may be absent or replaced by alternative isoforms.

Earlier work suggested that callus and latex have different repertoires of bioactive proteins, but the proteins underlying those activities were not characterized. The study investigated whether callus cultures of two laticiferous species reproduce the antifungal defense system previously described in their latex.

The approach combined histological analyses, antifungal assays, biochemical characterization, immunological detection, and PCR analysis of some pathogenesis-related proteins. The results associate antifungal activity with peptidases and osmotin-related proteins, while showing that callus and latex contain different tissue-specific protein isoforms.

The study therefore connects key antifungal protein expression with tissue differentiation in latex plants. Hypocotyl and cotyledon segments from both species were cultured on half-strength MS medium with sucrose, agar, one-naphthaleneacetic acid, and kinetin for callus induction.

The cultures were maintained in complete darkness at twenty-five plus or minus two degrees Celsius, and the calli were harvested after four weeks for histological, biochemical, and biological analyses. Histological analyses were performed exclusively on Cryptostegia grandiflora samples because the corresponding analyses for Calotropis procera had previously been reported.

Cotyledons, hypocotyls, and calli were fixed in Karnovsky’s solution for forty-eight hours, then dehydrated, infiltrated, and sectioned at five micrometers. The sections were stained with Astra blue and safranin or toluidine blue, briefly counterstained with basic fuchsin, mounted, and examined under a Leica microscope.

Calli from both species were homogenized in sodium chloride containing ascorbic acid, stirred for ninety minutes at four degrees Celsius, and centrifuged. Proteins were precipitated with ammonium sulfate and dialyzed against distilled water for forty-eight hours to remove residual salts and other low-molecular-weight compounds.

The dialyzed fractions were lyophilized and stored at twenty-five degrees Celsius; the callus fractions were designated CPCg for C. grandiflora and CPCp for C. procera. Antifungal activity was evaluated against Fusarium solani, Fusarium oxysporum, Colletotrichum lindemuthianum, and Colletotrichum gloeosporioides.

The fungi were maintained on Sabouraud Dextrose Agar at twenty-seven plus or minus two degrees Celsius in the dark and were subcultured monthly. Spore suspensions were prepared from fifteen-day-old cultures, filtered through sterile nylon mesh, counted with a Neubauer chamber, and adjusted to two times ten to the fifth spores per milliliter.

For spore germination assays, spore suspension was mixed with protein samples at five milligrams per milliliter and incubated at twenty-seven degrees Celsius in the dark. Germination was assessed microscopically after twenty-four hours. For mycelial growth inhibition, spores were incubated in yeast peptone dextrose medium before protein samples were added, and growth was monitored by turbidity at six hundred twenty nanometers for forty-eight hours.

Sodium acetate buffer and hydrogen peroxide served as negative and positive controls, respectively. All in vitro antifungal and enzymatic activity assays used three independent biological replicates, each comprising four technical replicates.

The data were analyzed with GraphPad Prism version six point zero. Statistical significance was assessed using one-way analysis of variance followed by Tukey’s post hoc test, with a probability below zero point zero five considered significant. Laticifers were detected in both cotyledons and hypocotyls of Cryptostegia grandiflora.

In cotyledons, they appeared in the midrib and mesophyll, arranged longitudinally to the leaf veins and predominantly associated with vascular bundles in the midrib. Their dense luminal contents were consistent with secretory activity, and hypocotyl laticifers occurred in the cortical region with rounded or elongated forms.

Laticifers were not detected in Cryptostegia grandiflora calli, which contained two distinct cellular populations. Inner callus cells were densely packed and showed features indicative of intense mitotic activity, while surface cells were highly vacuolated with large intercellular spaces characteristic of friable callus.

Calotropis procera callus cultures also lacked detectable laticifers, so subsequent biochemical and biological assays used callus protein extracts from both species. The study compared callus protein fractions from both species with latex protein fractions from both species for antifungal activity.

Calotropis procera callus proteins strongly inhibited mycelial growth, with inhibition rates of seventy-five point five percent for Fusarium solani, eighty-eight point eight percent for Fusarium oxysporum, ninety-three point seven percent for Colletotrichum lindemuthianum, and eighty point two percent for Colletotrichum gloeosporioides.

Cryptostegia grandiflora callus proteins did not inhibit mycelial growth of any evaluated fungus, whereas latex fractions from both species were active against all tested fungi. The same pattern appeared in spore germination: CPCp inhibited all four fungi, while CPCg showed no inhibition.

The mechanistic assays focused on whether callus-derived fractions disrupted fungal plasma membranes and induced oxidative stress. Colletotrichum gloeosporioides was selected as the representative species for these analyses. CPCp-treated spores showed intense propidium iodide fluorescence, indicating plasma-membrane damage and increased membrane permeability, while CPCg caused no detectable membrane damage.

DAB staining showed little hydrogen peroxide accumulation with CPCg, but intense reddish-brown staining with CPCp, indicating oxidative stress associated with reactive oxygen species. In panel A, the CPCp fluorescence image contains several bright spore-shaped signals against a dark background, whereas the corresponding CPCg image remains nearly uniformly dark, resembling the control.

In panel B, the CPCp field shows visibly darker reddish-brown spores than the pale control and CPCg fields, providing a clear visual contrast in DAB staining. The Cryptostegia grandiflora callus system contained both cysteine and serine peptidase inhibitors, based on inhibition of papain and trypsin.

CPCp inhibited papain activity by sixty-one point six percent but did not affect trypsin activity, suggesting no detectable serine peptidase inhibitors in the Calotropis procera callus fraction. Chitinolytic activity was one hundred sixty-six nanokatals per microgram of protein in the latex fraction of Cryptostegia grandiflora and one hundred forty-six in its callus fraction.

The Calotropis procera latex fraction showed lower activity at eighty-one nanokatals per microgram of protein, while its callus fraction reached three hundred fifty-eight. A purified chitinase fraction from Calotropis procera latex was tested to see whether chitinases contributed to antifungal activity.

That purified fraction did not inhibit the growth of any of the four phytopathogenic fungi tested. The study also investigated osmotins previously purified from the latex of Calotropis procera and Cryptostegia grandiflora. Because the osmotins were structurally similar, antibodies raised against CpOsm were used to detect osmotins in callus protein fractions.

ELISA showed strong recognition of CpOsm in LPCp but only weak recognition in CPCp, suggesting that CPCp contains an osmotin-related protein differing from CpOsm. Weak recognition also occurred in LPCg, while no antibody recognition was detected in CPCg; dot blot assays confirmed this pattern.

Proteolytic activity was consistently higher in Calotropis procera samples than in Cryptostegia grandiflora, especially when BANA was used, suggesting a predominance of cysteine peptidases in CPCp. Low activity with BAPNA suggested the absence or very low abundance of serine peptidases in the analyzed fractions.

Zymography detected clear proteolytic bands in CPCp but no proteolytic activity in Cryptostegia grandiflora callus extracts. The CPCp proteolytic profile differed from LPCp, suggesting that callus peptidases are distinct from those found in latex. To test whether cysteine peptidases contributed to antifungal activity, all protein samples were treated with iodoacetamide, a specific and irreversible inhibitor of cysteine peptidases.

After iodoacetamide treatment, all fractions almost completely lost their proteolytic activity. Antifungal activity was also partially abolished, by eighty to eighty-five percent, in previously active fractions including Calotropis procera latex and callus proteins and Cryptostegia grandiflora latex proteins.

These findings support a major contribution of cysteine peptidase activity to the antifungal effects of these protein fractions. RT-PCR analyses used primers targeting the latex cysteine peptidase CpPep and osmotin CpOsm genes to investigate transcript-level expression in non-laticiferous callus tissue.

The corresponding peptidase and osmotin transcripts were detected in latex, whereas no amplification products were detected in the corresponding callus samples under the tested conditions. This transcriptional pattern was consistent with the biochemical and immunological profiles of the corresponding protein fractions.

In the left EF1α panel, both CPCal and CPLa show similarly positioned bands, confirming comparable reference-gene amplification. In contrast, the CpOsm and CpPep panels show visible bands only in the CPLa lanes, while the corresponding CPCal lanes remain blank. The study concludes that Calotropis procera callus-derived proteins retain antifungal activity associated with peptidases and osmotins, whereas Cryptostegia grandiflora callus proteins lack that activity.

C. procera callus retained antifungal activity through cysteine peptidases and osmotins, while those proteins differed from latex forms. The result links defense chemistry to tissue specialization.