Skip to main content
NCBI home page
Plants logoLink to Plants
. 2026 Aug 20;15(16):2524. doi: 10.3390/plants15162524

Comparative Biology Research on the Reproductive Organs Between Physalis pubescens L. and Solanum lycopersicum var. cerasiforme

Xuemeng Shan 1,2, Xuechao Feng 1,2, Lida Zhang 1,2, Lingxia Zhao 1,2,*
Editor: Paolo Costantino
PMCID: PMC13516136  PMID: 42654927

Abstract

The post-anthesis sepal inflation forming a Chinese lantern in Physalis pubescens represents a striking morphological novelty, whereas in the related genus Solanum lycopersicum var. cerasiforme, the sepals remain non-enclosing, providing an ideal comparative system to study reproductive organ divergence. This study aimed to systematically compare the reproductive development between Physalis pubescens L. and Solanum lycopersicum var. cerasiforme. We integrated phylogenetic analysis, light and scanning electron microscopy, semi-thin sectioning, and quantitative RT-qPCR analysis of six MADS-box genes. Phylogenetic analysis placed P. pubescens in a clade with P. alkekengi (L.) and P. ixocarpa (Brot. ex Hornem.), distinct from tomato. Floral organs differed markedly in petal color, anther morphology, and dehiscence type. Microspore development was delayed before the tetrad stage but accelerated thereafter in P. pubescens; S. lycopersicum anthers exhibited pronounced connective tissue proliferation absent in P. pubescens. P. pubescens sepals expanded 6.92-fold by 17 days post anthesis and enclosed the fruit, while S. lycopersicum sepals grew minimally. Sepal expansion was driven by inner epidermal cell enlargement and intercellular space formation, producing a hollow structure with a trichome-free inner epidermis. Expression of six MADS-box genes exhibited diversity: PfMPF2 and PfAGL1 were upregulated during sepal growth, PfMPF3, PfSEP1 and PfSEP3 exhibited a high–low–high pattern with minima at peak growth, and PfAGL6 declined. These findings reveal that coordinated epidermal cell expansion, parenchyma cavity formation, and a dynamic MADS-box gene network govern the inflated sepal syndrome, and highlight key divergences in anther morphogenesis, providing a cellular and molecular framework for reproductive evolution in Solanaceae.

Keywords: inflated sepal, fruit development, anther connective tissue, sepal development, microspore development, MADS-box genes, comparative biology

1. Introduction

Physalis pubescens L. (downy groundcherry), native to South America, is a semi-cultivated fruit crop now grown in many temperate and subtropical regions, and has become an economically important fruit in Northeast China [1,2]. The plant produces globose yellow berries prized for their sweet–tart flavor and rich nutritional value. Consistent with its traditional medicinal use, extracts and isolated compounds from Physalis species have demonstrated diverse pharmacological activities. For example, physalin E, a secosteroid purified from Physalis angulata L., inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB (NF-κB) activation in RAW 264.7 macrophages at concentrations of 10–30 μM in vitro [3], supporting the anti-inflammatory properties attributed to the genus. The most striking feature of P. pubescens is the post-anthesis behavior of its sepal. After pollination, the sepals undergo dramatic enlargement to form a papery, lantern-like structure that completely encloses the fruit, a phenomenon termed inflated sepal syndrome [4,5]. The inflated sepal syndrome serves as a physical barrier against herbivory and mechanical damage and has evolved in multiple solanaceous genera [6,7,8]. In sharp contrast, the sepal of tomato (S. lycopersicum), a close relative and model solanaceous plant, remains small and non-enclosed throughout development, providing an ideal comparative system [9,10,11]. Beyond sepal differences, the two species diverge in multiple reproductive traits: for example, P. pubescens has pale yellow petals with a purple eye-like pattern and purple anthers with fertile apical necks and dorsal dehiscence, whereas S. lycopersicum has yellow lanceolate petals and apically sterile, ventrally dehiscent anthers. These species also differ in microspore developmental timing, anther connective tissue proliferation, and fruit maturation patterns [12,13]. The temporal shift in microspore development—delayed before meiosis but accelerated after tetrad release in Physalis—may reflect divergent regulation of conserved developmental programs, such as the AMS and MYB-dependent networks controlling pollen differentiation [14]. In contrast, the dramatic connective tissue proliferation observed in S. lycopersicum anthers, which is completely absent in P. pubescens, has been linked to the formation of the anther cone and may represent an evolutionary adaptation to buzz-pollination [15]. However, comparative analyses integrating these traits remain unexplored.

The inflated sepal syndrome is regulated by a network of MADS (MCM1, AGAMOUS, DEFICIENS, and SRF)-box transcription factors [16,17,18]. Heterotopic expression of PfMPF2 in the sepal is the key trigger of inflated sepal syndrome [4]. Mutations in the PfMPF2 promoter abolish repression by the A-class protein MPF3, enabling sepal-specific expression [19]. PfMPF2 interacts with SEPALLATA proteins Pf-SEP1, Pf-SEP3, and PfAGL6 to form regulatory complexes that presumably activate downstream genes involved in cell division and cell wall remodeling, thereby driving the rapid post-anthesis calyx expansion [20]. In S. lycopersicum, the orthologs of these genes show markedly different expression patterns and the corresponding proteins may not assemble into functionally equivalent complexes, failing to induce sepal inflation [17,21,22,23]. Despite these advances, how the expression of positive and negative regulators is coordinated to control the timing and magnitude of sepal expansion, and how broader reproductive traits differ between these two species, remain poorly understood.

However, the cellular basis of sepal inflation and the coordinated regulation of MADS-box genes during this process remain poorly understood. Moreover, how anther developmental programs differ between P. pubescens and S. lycopersicum has not been systematically characterized. The aim of this study was to elucidate the cellular and molecular basis of sepal inflation and to identify key divergences in reproductive development between P. pubescens and S. lycopersicum. To this end, we integrated phylogenetic analysis, morphological characterization of floral organs, fruits and microspore development, semi-thin sectioning, scanning electron microscopy, and quantitative expression analysis of six key MADS-box genes throughout reproductive development. Our results establish the cellular and molecular framework for reproductive organs between P. pubescens and S. lycopersicum and reveal novel divergences in reproductive development between these two solanaceous species.

2. Results

2.1. Phylogenetic Analysis of P. pubescens Within the Solanaceae

To determine the phylogenetic position of P. pubescens within the Solanaceae and to provide a genetic framework for understanding its unique sepal development, we performed phylogenetic analysis based on three widely used DNA markers: the Nuclear ribosome deoxyribonucleic acid internal transcribed spacer (nrDNA-ITS), the Granule-bound starch synthase (GBSSI), and the Transfer ribonucleic acid leucine-transfer ribonucleic acid phenylalanine (trnL-F). A total of 18 accessions representing 14 species from five genera of Solanaceae, together with Arabidopsis thaliana (L.) as an outgroup, were included in this study. Genomic DNA was extracted and the target DNA fragments were amplified by PCR using specific primers (Supplementary Figure S1). Genetic distances among the 18 accessions were calculated based on the combined sequence data of the three markers. The results showed that P. pubescens had the smallest genetic distance to P. ixocarpa and P. alkekengi, indicating the closest relationship within the genus, and the highest genetic distance to A. thaliana, confirming its distant relationship to the outgroup. Phylogenetic trees constructed using the neighbor-joining method consistently placed P. pubescens in a well-supported clade with P. ixocarpa and P. alkekengi, clearly separated from the tomato subgroup and other subgroups (Figure 1). This phylogenetic distance provides a meaningful evolutionary framework for the comparative analysis of sepal development between P. pubescens and tomato.

Figure 1.

Figure 1

Open in a new tab

The phylogenetic tree based on nrDNA-ITS, GBSSI and trnL-F. The P. pubescens subgroup was marked with a blue frame; the tomato subgroup was marked with a green frame; the S. nigrum was marked with a pink frame; the N. benthamiana was marked with a purple frame; The Arabidopsis was marked with a gray frame. Bootstrap values (1000 replicates) are indicated at the nodes.

2.2. Plant and Flower Morphology of P. pubescens and S. lycopersicum

To characterize the morphological differences between P. pubescens and S. lycopersicum, we compared their overall plant architecture, flower, pollen staining and pollen shape. P. pubescens exhibited a semi-trailing, determinate growth habit with simple ovate leaves, whereas S. lycopersicum displayed an indeterminate, sprawling architecture with compound pinnate leaves (Figure 2A,C). The flowers of P. pubescens were smaller than those of S. lycopersicum, with pale yellow petals forming a pentagonal corolla that bore a purple eye-like pattern at the throat. In contrast, S. lycopersicum cerasiforme produced yellow, lanceolate petals without pigmentation (Figure 2B,D).

Figure 2.

Figure 2

Open in a new tab

Plant and flower of P. pubescens and S. lycopersicum. (A): The plant of P. pubescens, bars = 3 cm; (B): the flower of P. pubescens, bars = 1 cm; (C): the plant of S. lycopersicum, bars = 3 cm; (D): the flower of S. lycopersicum, bars = 1 cm.

Acetocarmine staining showed that P. pubescens and S. lycopersicum pollen had high fertility (Figure 3A,C, Supplementary Figure S2). Pollen grains of both species were spherical; however, P. pubescens pollen was slightly larger in diameter than that of S. lycopersicum (Figure 3B,D).

Figure 3.

Figure 3

Open in a new tab

The pollen staining and pollen shape of P. pubescens and S. lycopersicum. (A): Acetocarmine-stained pollen of P. pubescens. Fertile grains (black arrows) are round and deeply stained; sterile grains (white arrows) are shrunken and stain weakly, bars = 50 μm; (B): the pollen of P. pubescens, bars = 5 μm; (C): acetocarmine-stained pollen of S. lycopersicum. Fertile grains (black arrows) are round and deeply stained; sterile grains (white arrows) are shrunken and stain weakly, bars = 50 μm; (D): the pollen of S. lycopersicum, bars = 5 μm.

2.3. Floral Organ Morphology and Microspore Development in P. pubescens and S. lycopersicum

We further compared floral bud development from −11 dpa to anthesis (0 dpa) in the two species. At −11 dpa, buds of both species were yellowish and tightly closed, but their subsequent developmental diverged distinctly. P. pubescens buds remained yellow and tightly closed until approximately −5 dpa, whereas buds of S. lycopersicum began to turn green at −10 dpa and started to open at approximately −9 dpa. At anthesis, the pale-yellow petals of P. pubescens displayed a characteristic purple eye-like pattern at the throat, and the pale purple anthers possessed fertile apical necks and dehisced via dorsal sutures. In contrast, S. lycopersicum had yellow, lanceolate petals without pigmentation patterning, and its yellow anthers were apically sterile, laterally connate, forming an anther cone, and dehisced ventrally (Figure 4A,C).

Figure 4.

Figure 4

Open in a new tab

Floral bud development and microsporogenesis in P. pubescens and S. lycopersicum from −11 dpa to 0 dpa. (A): The flower bud development of P. pubescens, bars = 1.0 cm; (B): semi-thin sections of anther development in P. pubescens (−11 dpa, −10 dpa, −9 dpa, −8 dpa, −7 dpa, −6 dpa, −5 dpa, −4 dpa, −3 dpa, −2 dpa, −1 dpa, 0 dpa); bars = 50 μm. At −11 dpa, sporogenous cells were well organized and the tapetum was distinguishable; tetrads appear at −8 dpa; microspores are released from tetrads by −7 dpa and subsequently mature into pollen grains without connective tissue proliferation. (C): The flower bud development of S. lycopersicum, bars = 1.0 cm; (D): semi-thin sections of anther development in S. lycopersicum (−11 dpa, −10 dpa, −9 dpa, −8 dpa, −7 dpa, −6 dpa, −5 dpa, −4 dpa, −3 dpa, −2 dpa, −1 dpa, 0 dpa); bars = 50 μm. At −11 dpa, the locules were not yet U-shaped and the tapetum was indistinct; tetrads were already present at −9 dpa; pronounced connective tissue proliferation begins at −7 dpa and progressively occupies more than half of the locule volume by anthesis (0 dpa). C, connective tissue; E, epidermis; En, endothecium; ML, middle layer; MMC, microspore mother cells; MSp, microspores; PG, pollen grains; Sm, septum; Sp, sporogenous cells; StR, stomium region; T, tapetum; Tds, tetrads; V, vascular bundle; Va, vacuoles.

Semi-thin transverse sections of anthers revealed that microspore development in both species could be divided into eleven distinct stages (Figure 4B,D). At the U-shaped sporogenous cell stage (−11 dpa), a single layer of sporogenous cells was already well organized in P. pubescens, whereas in S. lycopersicum, the four locules had not yet adopted a U-shaped configuration and the tapetum was indistinguishable. Before the tetrad stage (−10 to −9 dpa), microspore development in P. pubescens was delayed relative to S. lycopersicum: at −9 dpa, S. lycopersicum had already entered the tetrad stage, whereas P. pubescens was still at the microspore mother cell stage. After the release of uninucleate microspores from tetrads (after −8 dpa), however, development in P. pubescens gradually overtook that in S. lycopersicum, and pollen maturation and shedding occurred slightly earlier. The most striking cellular divergence was the abnormal proliferation of the connective tissue between the two locules of a single theca in S. lycopersicum, which began at −7 dpa, progressively protruded into the locular cavity, and occupied more than half of the locule volume at anthesis, a feature that is completely absent in P. pubescens (Figure 4). Collectively, these results demonstrate that the two solanaceous species show distinct developmental programs during anther morphogenesis.

2.4. Comparative Morphology and Post Anthesis Sepal Development in P. pubescens and S. lycopersicum

To characterize the differences in sepal development between P. pubescens and S. lycopersicum, we compared their sepal development morphology and sepal growth dynamics after anthesis. The post anthesis developmental patterns of the sepal were markedly different between P. pubescens and S. lycopersicum. The sepal of P. pubescens grew rapidly, while the S. lycopersicum grew relatively slowly (Figure 5I,R). In addition, the midrib of the P. pubescens sepal protruded to form prominent ridges, while it was not obvious in S. lycopersicum (Figure 5A–H,J–Q). At anthesis (0 dpa), the sepal of P. pubescens consisted of five sepals with a bell-shaped structure, which was 0.52 ± 0.05 cm in length and 0.21 ± 0.01 cm in width. The sepal of S. lycopersicum was deeply split, with a length of 0.86 ± 0.13 cm and a width of 0.14 ± 0.01 cm (Figure 5I,R). After pollination, the sepal of P. pubescens expanded rapidly, and the vein area protruded to form obvious ridges, and finally developed into a completely closed, lantern-shaped structure, which wrapped the whole developing fruit. In contrast, the growth of S. lycopersicum sepal after flowering was very limited, and only a small inverted structure remained, which adhered to the basal end of the fruit and did not form a package. To precisely quantify the differential growth patterns, we measured sepal length and width at 2-day intervals from 1 dpa to 17 dpa. The sepal of P. pubescens displayed a pronounced sigmoidal growth curve, characterized by a relatively slow initial phase (1–5 dpa), followed by a rapid exponential growth phase (5–13 dpa), and finally reaching a plateau (13–17 dpa). By 17 dpa, the P. pubescens sepal had attained a length of 3.60 ± 0.07 cm and a width of 1.23 ± 0.09 cm, representing a 6.92-fold increase in length and a 5.86-fold increase in width relative to their values at anthesis. In contrast, the S. lycopersicum sepal exhibited only marginal growth throughout the same period, reaching a final length of 1.87 ± 0.13 cm and width of 0.27 ± 0.01 cm at 17 dpa, corresponding to merely 2.17-fold and 1.93-fold increases, respectively.

Figure 5.

Figure 5

Open in a new tab

Comparative sepal morphology and growth dynamics of P. pubescens and S. lycopersicum. (A–H): The sepal development (1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa 11 dpa, 13 dpa, 15 dpa) of P. pubescens, bars = 5 mm; (I): the sepal length and width of P. pubescens; (J–Q): the sepal development (1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa 11 dpa, 13 dpa, 15 dpa) of S. lycopersicum, bars = 5 mm; (R): the sepal length and width of S. lycopersicum.

These results demonstrate that the formation of the Chinese lantern phenotype in P. pubescens is primarily driven by a phase of accelerated sepal expansion initiated around 5–7 dpa, a developmental program that was largely absent in the sepal of S. lycopersicum. This pronounced difference in post-anthesis growth trajectory provided a quantitative framework for investigating the underlying cellular and molecular mechanisms of inflated sepal syndrome.

2.5. The Morphological Comparison of Different Fruit Development Stages in P. pubescens and S. lycopersicum

The fruit development of P. pubescens is structurally characterized by the coordinated growth of the accrescent sepal and the inner berry. Following anthesis and initial fruit set, the sepal rapidly closes and undergoes a dramatic, lantern-like expansion synchronized with berry enlargement, completely enveloping the developing fruit. Throughout the developmental trajectory, the berry transitions from an early whitish-green to a uniform bright green, eventually turning a bright golden yellow at full maturity; simultaneously, the outer sepal gradually de-greens, desiccates, and ultimately transforms into a brown, paper-like husk (Figure 6A–J).

Figure 6.

Figure 6

Open in a new tab

The morphological changes during fruit development and ripening in P. pubescens and S. lycopersicum. (A–J): The developmental stages (1 dpa, 5 dpa, 9 dpa, 13 dpa, 17 dpa, 21 dpa, 23 dpa, 25 dpa, break ripen stage, ripen stage) of P. pubescens from anthesis to full maturity; (K–T): the developmental stages (1 dpa, 5 dpa, 9 dpa, 13 dpa, 17 dpa, 21 dpa, 23 dpa, 25 dpa, break ripen stage, ripen stage) of S. lycopersicum from anthesis to full maturity; bars = 1.0 cm.

Distinct from P. pubescens, S. lycopersicum fruit development exhibits naked berry growth accompanied by substantial volume expansion. Following petal senescence, abscission, and successful fruit set, the young fruit emerges from the basal persistent green sepals and enters a rapid enlargement phase, gradually shaping into a smooth, plump, subglobose form. The pericarp maintains a dark or light green coloration throughout the immature green stages until entering the breaker stage upon ripening initiation, transitioning through yellowish-green before ultimately shifting into a uniform, highly saturated bright orange at the fully ripe stage (Figure 6K–T).

2.6. The Difference in Tissue Structure of Sepal in Cross Section

To further investigate the microstructure difference between P. pubescens and S. lycopersicum sepal, we conducted a semi-thin section experiment. The results showed that the sepal of P. pubescens and S. lycopersicum were composed of inner and outer epidermis, trichomes, parenchyma cells and vascular bundles; however, the epidermis structure was not observed in the inner epidermis of P. pubescens (Figure 7). The number of sepal cells in P. pubescens was less than that of S. lycopersicum, and the size of parenchyma cells increased from 3 dpa, accompanied by a marked increase in intercellular spaces, and by 15 dpa, the parenchyma tissue had become largely hollow. However, the parenchyma cells of S. lycopersicum were arranged closely, and the parenchyma cells grow rapidly from 9 dpa, and intercellular spaces only became evident at this stage. Different from other tissues dyed light blue, vascular bundle tissues were dyed with toluidine blue to form small and dense oval tissues. The inner and outer sepals of the two species were composed of monolayer cells, which are arranged neatly and interconnected tightly.

Figure 7.

Figure 7

Open in a new tab

Semi-thin sections of sepal cells in P. pubescens and S. lycopersicum. (A–H): Semi-thin sections of sepal cells of P. pubescens in sepal development of 1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa 11 dpa, 13 dpa,15 dpa ((A): 1 dpa, (B): 3 dpa, (C): 5 dpa, (D): 7 dpa, (E): 9 dpa, (F): 11 dpa, (G): 13 dpa, (H): 15 dpa); (I–P): semi-thin sections of sepal cells of S. lycopersicum in sepal development of 1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa 11 dpa, 13 dpa,15 dpa ((I): 1 dpa, (J): 3 dpa, (K): 5 dpa, (L): 7 dpa, (M): 9 dpa, (N): 11 dpa, (O): 13 dpa, (P): 15 dpa), bars = 100 μm. Semi-thin sections of sepals stained with toluidine blue O. Cg, cell gap; Pc, parenchyma cells; Tr, trichome; Vb, vascular bundle.

2.7. The Difference in Epidermal Cells of Sepal

Based on the results of the semi-thin section, we conducted a scanning electron microscope. The results showed that there was no difference between the outer epidermal cells of P. pubescens sepals and S. lycopersicum, and they all have trichomes and a stomatal structure. At 1 dpa, the outer epidermal cells of P. pubescens were smaller than those of S. lycopersicum. However, by 7 dpa, the outer epidermal cells of P. pubescens sepals grew faster than those of S. lycopersicum. From the 11 dpa, the outer epidermal cells of P. pubescens sepals became larger than those of S. lycopersicum, with the cell walls becoming thinner and the cell shape transitioning from oval to irregular (Figure 8).

Figure 8.

Figure 8

Open in a new tab

The cell profiles of sepal outer epidermis of P. pubescens and S. lycopersicum. (A–H): The cell profiles of sepal outer epidermis of P. pubescens in sepal development of 1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa 11 dpa, 13 dpa, 15 dpa ((A): 1 dpa, (B): 3 dpa, (C): 5 dpa, (D): 7 dpa, (E): 9 dpa, (F): 11 dpa, (G): 13 dpa, (H): 15 dpa); (I–P): the cell profiles of sepal outer epidermis of S. lycopersicum in sepal development of 1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa, 11 dpa, 13 dpa, 15 dpa ((I): 1 dpa, (J): 3 dpa, (K): 5 dpa, (L): 7 dpa, (M): 9 dpa, (N): 11 dpa, (O): 13 dpa, (P): 15 dpa), bars = 25 μm. St, stomata; Tr, trichome.

The inner epidermis of S. lycopersicum sepals was similar to its own outer epidermis with a stomata and epidermis structure, but the epidermis of P. pubescens sepals was not observed (Figure 9). The size of epidermal cells in P. pubescens was smaller than that of S. lycopersicum at 1 dpa. The inner epidermal cells are slightly larger. However, at 7 dpa, the epidermis of P. pubescens sepals, especially the inner epidermis, grew faster than that of S. lycopersicum (Table 1). From the 11 dpa, the inner and outer epidermis cells of P. pubescens sepals were larger than those of S. lycopersicum, and the cell wall became thinner and the cell shape became irregular from oval (Figure 8 and Figure 9). The microstructure suggested that the rapid expansion of sepal of P. pubescens was mainly caused by the increase in epidermal cell size and parenchyma cell gap of sepals.

Figure 9.

Figure 9

Open in a new tab

The cell profiles of sepal inner epidermis of P. pubescens and S. lycopersicum. (A–H): The cell profiles of sepal outer epidermis of P. pubescens in sepal development of 1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa 11 dpa, 13 dpa, 15 dpa ((A): 1 dpa, (B): 3 dpa, (C): 5 dpa, (D): 7 dpa, (E): 9 dpa, (F): 11 dpa, (G): 13 dpa, (H): 15 dpa); (I–P): the cell profiles of sepal outer epidermis of S. lycopersicum in sepal development of 1 dpa, 3 dpa, 5 dpa, 7 dpa, 9 dpa, 11 dpa, 13 dpa, 15 dpa ((I): 1 dpa, (J): 3 dpa, (K): 5 dpa, (L): 7 dpa, (M): 9 dpa, (N): 11 dpa, (O): 13 dpa, (P): 15 dpa), bars = 25 μm. St, stomata; Tr, trichome.

Table 1.

The cell size of sepal epidermis of P. pubescens and S. lycopersicum (μm2).

dpa (P. pubescens) (S. lycopersicum)
Outer Epidermis Inner Epidermis Outer Epidermis Inner Epidermis
1 287 ± 59 500 ± 73 789 ± 162 365 ± 64
3 350 ± 68 500 ± 69 855 ± 112 712 ± 103
5 339 ± 70 499 ± 39 859 ± 140 622 ± 160
7 374 ± 38 518 ± 83 814 ± 129 659 ± 44
9 935 ± 101 3560 ± 238 1483 ± 63 736 ± 143
11 2249 ± 395 3508 ± 682 1609 ± 293 646 ± 116
13 2486 ± 303 3441 ± 707 1716 ± 282 730 ± 165
15 2607 ± 413 3838 ± 600 2067 ± 150 902 ± 160
Open in a new tab

2.8. Expression Patterns of Genes Related to Sepal Development

To investigate the molecular regulation of sepal development, we analyzed the expression patterns of six MADS-box genes related to sepal development in different organs. PfMPF2 (MADS box gene 2 from P. pubescens) was predominantly expressed in vegetative organs of P. pubescens, especially in roots, whereas its S. lycopersicum ortholog SlSVP (Short Vegetative Phase) was mainly expressed in stems and leaves. PfMPF3 (MADS box gene 3 from P. pubescens), PfSEP1 (Physalis floridana SEPALLATA1), and PfSEP3 (Physalis floridana SEPALLATA3) were highly expressed in floral organs of P. pubescens, while their S. lycopersicum orthologs SlMADS-MC (MACROCALYX), SlTM29 (Tomato MADS box gene No.29), and SlTM5 (Tomato MADS box gene No.5) showed distinct organ specific expression patterns. PfAGL1 (Physalis floridana AGAMOUS-like 1) and its ortholog SlTAGL1 (Tomato AGAMOUS-like 1) were both specifically expressed in pistils and fruits. PfAGL6 (Physalis floridana AGAMOUS-like 6) was expressed in vegetative organs, whereas SlAGL6 (S. lycopersicum AGAMOUS-like 6) was expressed in floral organs of S. lycopersicum (Figure 10). These results indicate that the organ specific expression of these MADS-box genes has substantially diverged between the two species, which may underlie their distinct sepal developmental programs.

Figure 10.

Figure 10

Open in a new tab

The expression patterns in different organs of genes related to sepal development in P. pubescens and S. lycopersicum. (A): PfMPF2, MADX box gene 2 from P. pubescens; SlSVP, Short Vegetative Phase; (B): PfMPF3, MADS box gene 3 from P. pubescens; SlMADS-MC, MACROCALYX; (C): PfSEP1, Physalis floridana SEPALLATA1; SlTM29, Tomato MADS box gene No.29; (D): PfSEP3, Physalis floridana SEPALLATA3; SlTM5, Tomato MADS box gene No.5; (E): PfAGL1, Physalis floridana AGAMOUS-like 1; SlTAGL1, Tomato AGAMOUS-like 1; (F): PfAGL6, Physalis floridana AGAMOUS-like 6; SlAGL6, Solanum lycopersicum AGAMOUS-like 6. The capital letters indicate statistical significance at p < 0.01 as determined by Duncan’s test.

Throughout sepal development, these genes exhibited divergent temporal expression dynamics between the two species. PfMPF2 expression continuously increased during sepal development, while SlSVP declined in S. lycopersicum sepals. PfMPF3, PfSEP1, and PfSEP3 displayed a high–low–high expression pattern, with expression minima at the period of fastest sepal growth (7 dpa) suggesting the negative regulatory roles; the orthologs genes of S. lycopersicum showed distinct expression patterns. PfAGL1 was significantly upregulated after 7 dpa, coinciding with rapid sepal expansion, whereas SlTAGL1 declined over the same period. The PfAGL6 and SlAGL6 both declined progressively during sepal development. These results indicate that the accelerated sepal growth in P. pubescens is temporally associated with the upregulation of positive regulators (PfMPF2, PfAGL1) and the transient downregulation of negative regulators (PfMPF3, PfSEP1, PfSEP3), while PfAGL6 showed a progressive decline, a pattern temporally associated with the later stages of sepal development (Figure 11).

Figure 11.

Figure 11

Open in a new tab

The expression patterns in different sepal development stages of genes related to sepal development in P. pubescens and S. lycopersicum. (A): PfMPF2, MADX box gene 2 from P. pubescens; SlSVP, Short Vegetative Phase; (B): PfMPF3, MADS box gene 3 from P. pubescens; SlMADS-MC, MACROCALYX; (C): PfSEP1, Physalis floridana SEPALLATA1; SlTM29, Tomato MADS box gene No.29; (D): PfSEP3, Physalis floridana SEPALLATA3; SlTM5, Tomato MADS box gene No.5; (E): PfAGL1, Physalis floridana AGAMOUS-like 1; SlTAGL1, Tomato AGAMOUS-like 1; (F): PfAGL6, Physalis floridana AGAMOUS-like 6; SlAGL6, Solanum lycopersicum AGAMOUS-like 6. The capital letters indicate statistical significance at p < 0.01, as determined by Duncan’s test.

3. Discussion

3.1. Phylogenetic Framework for Divergent Reproductive Development

The phylogenetic analysis consistently placed P. pubescens in a well-supported clade with P. alkekengi and P. ixocarpa, clearly separated from the tomato subgroup. This is consistent with previous molecular phylogenies of the Solanaceae [24,25] and provides an evolutionary context for the divergent reproductive traits we observed. The substantial genetic distance between P. pubescens and tomato supports the interpretation that their regulatory networks controlling reproductive development have undergone significant divergence since their last common ancestor. This phylogenetic separation emphasizes the value of comparative studies between these two genera for understanding how morphological novelties arise during evolution.

3.2. Cellular Basis of Inflated Sepal Formation in P. pubescens

Our comparative morphological and microstructural analyses demonstrated that the dramatic post-anthesis expansion of the P. pubescens sepal is driven jointly by the marked enlargement of epidermal cells (particularly the inner epidermis) and the gradual formation of intercellular spaces within the parenchyma, eventually giving rise to a hollow lantern-like structure by 15 dpa. This is consistent with the established view that the early growth of the P. pubescens sepal depends on PfMPF2-driven cell division [20]. However, our data further reveal that the subsequent rapid expansion phase relies primarily on cell vacuolization and the development of intercellular spaces. Rapid organ expansion typically requires cell wall loosening through the action of expansions and xyloglucan endotransglucosylase/hydrolases (XTHs) [26], whose expression and activity are under precise developmental and hormonal control [27]. The coordinated up- and down-regulation of MADS-box genes observed in this study may therefore directly or indirectly modulate these cell wall-related processes to drive the phase-specific growth of the lantern structure. Nevertheless, the specific regulatory mechanisms underlying these processes in the Physalis pubescens sepal remain to be clarified.

The inner epidermis of the P. pubescens sepal is completely devoid of trichomes, in sharp contrast to that of S. lycopersicum. Trichome initiation in plants is regulated by the MYB-bHLH-WD40 (MBW) transcriptional complex [28,29]. The polarized distribution of trichomes, with their absence on the inner epidermis of the Physalis pubescens sepal, may result from asymmetric regulation of this complex between the two epidermal layers. Whether this absence of trichomes relieves physical constraints on the outward expansion of inner epidermal cells, thereby promoting the inward curvature of the lantern, represents a morphological question worthy of further investigation.

3.3. Divergent Microspore Development and Anther Morphogenesis

Our detailed analysis of microspore development revealed a previously undocumented temporal divergence between these two solanaceous species. P. pubescens was delayed relative to S. lycopersicum before the tetrad stage but accelerated after tetrad release, with pollen maturation and shedding occurring slightly earlier. This pattern suggests evolutionary plasticity in the relative timing of early versus late microspore developmental phases. However, the most striking cellular difference was the dramatic proliferation of connective tissue between locules in tomato anthers, which progressively expanded to occupy more than half of the locular volume at anthesis. This feature was completely absent in P. pubescens. Connective tissue proliferation has been noted in other Solanum species [30] and may influence pollen dispersal dynamics, but its developmental regulation and functional significance remain poorly understood. The absence of this trait in P. pubescens identifies it as a derived character within the S. lycopersicum lineage and provides a unique opportunity for comparative functional genomic studies.

3.4. Dynamic Regulation of Sepal Growth by MADS-Box Genes

The expression analysis of six MADS-box genes revealed a striking temporal correlation between gene expression patterns and sepal growth dynamics. PfMPF2 expression continuously increased during sepal development, consistent with its established role as a master positive regulator of inflated sepal syndrome [4]. PfAGL1 was significantly upregulated after 7 dpa, precisely coinciding with the onset of rapid sepal expansion. Given that overexpression of SlTAGL1 (the tomato ortholog) can convert tomato sepals into fruit-like, expanded organs [9,31], the coincident upregulation of PfMPF2 and PfAGL1 suggests cooperative function, potentially through multimeric complexes bridged by SEPALLATA proteins to promote cell division and expansion.

The high–low–high expression pattern of PfMPF3, PfSEP1, and PfSEP3, with expression minima precisely at 7 dpa (the time of maximal growth rate), strongly connects these genes as growth repressors. PfMPF3, the A-class floral identity gene, is known to directly repress PfMPF2 in floral meristems [19]. The parallel downregulation of PfSEP1 and PfSEP3 suggests that repression of inflated sepal syndrome involves a broader network. SEP proteins are essential cofactors in nearly all floral quartets [32], and recent studies have highlighted their role beyond organ identity maintenance, namely in modulating organ size and growth rate through differential complex formation [33]. Thus, reduced availability during the rapid growth phase may shift the balance of MADS-box complexes from organ identity maintenance toward growth promotion. In S. lycopersicum, downregulation of SlTM29 leads to enlarged sepals [21], consistent with a growth-limiting role for SEP genes. The subsequent re-upregulation of PfSEP1 and PfSEP3 after 11 dpa coincides with growth deceleration and may serve as a feedback mechanism that prevents uncontrolled sepal expansion. PfAGL6 expression declined continuously in both species. SlAGL6 lineage genes control floral organ boundary formation and fusion [22,34], and the progressive decline in PfAGL6 coincided with the completion of sepal expansion, suggesting a potential role in the terminal differentiation rather than directly promoting convergence. Whether PfAGL6 downregulation contributes to the lantern shape through modulating organ boundary formation remains to be experimentally determined. Our findings thus establish a temporal framework in which the oscillating expression of SEP-class MADS-box genes tightly gates the growth-versus-differentiation balance during sepal inflation.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The seeds of P. pubescens were obtained by cutting fresh fruit from Harbin and air drying. The seeds of S. lycopersicum No.22 were stored at Shanghai Jiao Tong University, China. All plant lines were grown in a standard greenhouse at the Pujiang experimental base, Shanghai Jiao Tong University, Shanghai, China (121°30′10.89″ E, 31°3′5.20″ N, altitude 5 m).

4.2. Phylogenetic Analysis

Genomic DNA was extracted from young leaves of P. pubescens, tomato, pepper, eggplant and Arabidopsis by CTAB method [35], and PCR amplification and sequencing were carried out with corresponding primers described previously [36,37]. According to the sequencing results, phylogenetic tree was constructed by using MEGA7.0, neighbor joining (NJ) and 1000 repeated Bootstrap tests [38].

4.3. Botanical Characteristics Analysis

The P. pubescens and S. lycopersicum buds were taken every two days from 0 dpa to 17 dpa, sepal was peeled and photographed, and the length and width of sepal was measured with vernier caliper. The P. pubescens and S. lycopersicum buds were photographed every other day from −11 dpa to 0 dpa and fixed in FAA liquid (Sangon, Shanghai, China) for microspore development research. Photographs of the P. pubescens and S. lycopersicum fruits were taken every 4 days after flowering.

4.4. Pollen Vitality Analysis

Stamens of P. pubescens and S. lycopersicum were collected, and pollen was pricked with tweezers to take pollen on the glass slide and dropped 2–3 drops of 1% magenta acetocarmine staining solution. The pollen was incubated at 37 °C for 20 min to stain the pollen [39], and observed it under the microscope (Eclipse80i, Nikon, Tokyo, Japan). Fertile pollen grains were scored as those with round morphology and deep staining; sterile grains were scored as shrunken and weakly stained or unstained. At least 3 fields/20 grains were counted per sample.

4.5. Light Microscopy (LM) of Semi-Thin Sections

The P. pubescens and S. lycopersicum buds and sepals (cut into 3 × 2 mm2) at different development stages were immediately immersed in FAA fixed solution, and then stored in fresh FAA fixative at 4 °C [40]. The samples were dehydrated through a graded ethanol series (70%, 80%, 95%) for 30 min each, followed by 100% ethanol for 2 h. Dehydration was continued in a saturated solution of eosin in absolute ethanol for 2 h, after which the samples were infiltrated in a pre-infiltration solution (absolute ethanol: Basne Luqizd Technoovt 7100 = 1:1, v/v) for 2 h. The specimens were then placed in infiltration solution (1 g Hardener I dissolved in 100 mL Basne Luqizd Technoovt 7100) overnight at 25 °C. The samples were embedded in the embedding solution (hardener II: osmotic solution = v:v = 1:14) in an ice bath, heated at 48 °C for 10 min, and hardened in an oven at 60 °C for 48 h. Semi-thin sections (3 μm thick) were cut with a microtome (RM2265, LEICA, Wetzlar, Germany), stained with 1% toluidine blue O (TBO) (Sangon, Shanghai, China) for 40 s, rinsed with double-distilled water to remove excess stain, dried, and mounted with neutral balsam [41]. The sections were observed and photographed under a microscope (Eclipse 80i, Nikon, Tokyo, Japan).

4.6. Scanning Electron Microscopy (SEM)

The P. pubescens and S. lycopersicum sepals (cut into 3 × 2 mm2) at different development stages were immediately immersed in 2.5% glutaraldehyde (25% glutaraldehyde: 0.1 M phosphate buffer: double distilled water (v:v:v) = 1:5:4, pH = 7.3) for fixing. The samples were then placed under vacuum using a vacuum drying cabinet with a vacuum pump for 0.5 h, and then left at 4 °C for 12 h [42]. The samples were rinsed three times (for 15 min each) using 0.1 M phosphate buffer (pH = 7.2), immersed in 2% (w/v) osmium tetroxide for further fixing for 2 h, washed three times using 0.1 M phosphate buffer (15 min each time), and then dehydrated in an ethanol and acetone series as follows: 50% ethanol, 70% ethanol, 90% ethanol, 90% ethanol and acetone (v:v = 1:1), 90% acetone, each step for 15 min. Finally, the samples were dehydrated in acetone with shaking at 100 rpm (three times, 20 min each). After dehydration, the samples were immersed in acetone and epoxy resin 812 in different ratios (v:v = 1:1 and 1:2, for 1 h and overnight, respectively). The samples were then positioned on an embedding plate with 100% epoxy resin 812 for 7 h and kept at 60 °C for 48 h to form resin blocks. Thick sections (70 nm) were cut from the blocks using a Leica UC6-FC6 microtome with a diamond knife (Leica Microsystems Inc., Wetzlar, Germany) and stained with 2% lead citrate (w:v = 2%; Zhongjingkeyi Technology Co., Ltd., Beijing, China). The chromoplast ultrastructure was photographed using a scanning electron microscope (SEM; Verios G4, FEI, Hillsboro, OR, USA) at an accelerating voltage of 5–10 kV. Stomata and trichomes were identified and marked (St and Tr, respectively) on the SEM images.

4.7. Real-Time Quantitative PCR (RT-qPCR) Analysis

The sepals were sampled from P. pubescens and S. lycopersicum of the different stages for total RNA extraction using RNAprep pure Plant Kit (Tiangen, Beijing, China). Total RNA (1 μg) was used as a template to synthesize first-strand cDNA with the PrimeScriptTM RT Master Mix kit (TAKARA, Dalian, China) [43]. The cDNA library was diluted 50 times and used as a template for RT-qPCR analysis. RT-qPCR was performed on a Real-Time PCR System (QuantStudio 5, Thermo Fisher Scientific, Waltham, MA, USA) using SYBR Green chemistry (TAKARA, Dalian, China). The gene expression levels were calculated using the 2−ΔCT equation with ACTIN as an internal reference gene for normalization. RT-qPCR primers are listed in Supplementary Table S1.

4.8. Statistical Analysis

All experiments were performed with at least three biological replicates. Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 19. Differences between two groups were analyzed by Duncan’s multiple range test. Different letters indicate significant differences at p < 0.05 or p < 0.01.

5. Conclusions

In conclusion, this study presents analysis of the reproductive biology of P. pubescens and S. lycopersicum. Furthermore, we discovered that the inner epidermis of P. pubescens sepals was completely devoid of trichomes, in contrast to that of S. lycopersicum, which represented a novel morphological divergence potentially facilitating lantern formation by reducing physical constraints on inner epidermal cell expansion. Our results showed that the inflated sepal syndrome is driven by coordinated inner epidermal cell expansion, parenchyma intercellular space formation, and the dynamic regulation of a MADS-box gene network, with PfMPF2 and PfAGL1 acting as positive regulators and PfMPF3, PfSEP1, and PfSEP3 as negative regulators. We further identified significant differences in microspore development and anther morphogenesis, most notably the pronounced connective tissue proliferation in tomato anthers that is absent in P. pubescens. These findings provide a cellular and molecular framework and reveal key divergences in reproductive development within the Solanaceae. From an applied perspective, the genes and developmental modules identified in this study may serve as targets for breeding programs aimed at modifying fruit protection traits in solanaceous crops. The trichome-free inner epidermis of P. pubescens sepals represents a selectable morphological marker, and the MADS-box regulatory network controlling sepal inflation could be exploited to engineer physical barriers against pests and environmental stress in related species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants15162524/s1, Figure S1: Amplification of the target DNA fragment by PCR; Figure S2: The pollen fertility of P. pubescens and S. lycopersicum; Table S1: Primers used in this study.

plants-15-02524-s001.zip (372.6KB, zip)

Author Contributions

Conceptualization, X.S. and L.Z. (Lingxia Zhao); methodology, X.S.; validation, X.S. and X.F.; formal analysis, X.S.; investigation, X.S.; resources, X.S.; data curation, L.Z. (Lida Zhang); writing—original draft preparation, X.S.; writing—review and editing, X.F. and L.Z. (Lida Zhang); visualization, X.S.; supervision, X.F.; project administration, L.Z. (Lingxia Zhao); funding acquisition, L.Z. (Lingxia Zhao). All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was supported by National Natural Science Foundation of China (NSFC), grant number 32372694.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

References

  • 1.Sheikha A.E., Piombo G., Goli T., Montet D. Main composition of Physalis (Physalis pubescens L.) fruit juice from Egypt. Fruits. 2010;65:255–265. doi: 10.1051/fruits/2010021. [DOI] [Google Scholar]
  • 2.Yahia E.M. Woodhead Publishing Series in Food Science, Technology and Nutrition. Volume 2. Woodhead Publishing; Cambridge, UK: 2011. Postharvest biology and technology of tropical and subtropical fruits; pp. 1–532. [Google Scholar]
  • 3.Yang Y.J., Yi L., Wang Q., Xie B.B., Dong Y., Sha C.W. Anti-inflammatory effects of physalin E from Physalis angulata on lipopolysaccharide-stimulated RAW 264.7 cells through inhibition of NF-κB pathway. Immunopharmacol. Immunotoxicol. 2017;39:74–79. doi: 10.1080/08923973.2017.1282514. [DOI] [PubMed] [Google Scholar]
  • 4.He C.Y., Saedler H. Heterotopic expression of MPF2 is the key to the evolution of the Chinese lantern of Physalis, a morphological novelty in Solanaceae. Proc. Natl. Acad. Sci. USA. 2005;102:5779–5784. doi: 10.1073/pnas.0501877102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Whitson M., Manos P.S. Untangling Physalis (Solanaceae) from the Physaloids: A two-gene phylogeny of the Physalinae. Sys. Bot. 2005;30:216–230. doi: 10.1600/0363644053661841. [DOI] [Google Scholar]
  • 6.Pabón-Mora N., Litt A. Comparative anatomical and developmental analysis of dry and fleshy fruits of Solanaceae. Am. J. Bot. 2011;98:1415–1436. doi: 10.3732/ajb.1100097. [DOI] [PubMed] [Google Scholar]
  • 7.Wang L., Li J., Zhao J., He C.Y. Evolutionary developmental genetics of fruit morphological variation within the Solanaceae. Front. Plant Sci. 2015;6:248. doi: 10.3389/fpls.2015.00248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Pabón-Mora N., Wong G.K.S., Ambrose B.A. Evolution of fruit development genes in flowering plants. Front. Plant Sci. 2014;5:300. doi: 10.3389/fpls.2014.00300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Vrebalov J., Pan I.L., Arroyo A.J.M., McQuinn R., Chung M., Poole M., Rose J., Seymour G., Grandillo S., Giovannoni J., et al. Fleshy fruit expansion and ripening are regulated by the tomato SHATTERPROOF gene TAGL1. Plant Cell. 2009;21:3041–3062. doi: 10.1105/tpc.109.066936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Mao L., Begum D., Chuang H.W., Budiman M.A., Szymkowiak E.J., Irish E.E., Wing R.A. JOINTLESS is a MADS-box gene controlling tomato flower abscission zone development. Nature. 2000;406:910–913. doi: 10.1038/35022611. [DOI] [PubMed] [Google Scholar]
  • 11.de Martino G., Pan I., Emmanuel E., Levy A., Irish V.F. Functional analyses of two tomato APETALA3 genes demonstrate diversification in their roles in regulating floral development. Plant Cell. 2006;18:1833–1845. doi: 10.1105/tpc.106.042978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Pan I.L., McQuinn R., Giovannoni J.J., Irish V.F. Functional diversification of AGAMOUS lineage genes in regulating tomato flower and fruit development. J. Exp. Bot. 2010;61:1795–1806. doi: 10.1093/jxb/erq046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Valdenegro M., Fuentes L., Herrera R., Moya-León M.A. Changes in antioxidant capacity during development and ripening of goldenberry (Physalis peruviana L.) fruit and in response to 1-methylcyclopropene treatment. Postharvest Biol. Technol. 2012;67:110–117. doi: 10.1016/J.POSTHARVBIO.2011.12.021. [DOI] [Google Scholar]
  • 14.Gómez J.F., Talle B., Wilson Z.A. Anther and pollen development: A conserved developmental pathway. J. Integr. Plant Biol. 2015;57:876–891. doi: 10.1111/jipb.12425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.De Luca P.A., Vallejo-Marín M. What’s the ‘buzz’ about? The ecology and evolutionary significance of buzz-pollination. Curr. Opin. Plant Biol. 2013;16:429–435. doi: 10.1016/j.pbi.2013.05.002. [DOI] [PubMed] [Google Scholar]
  • 16.Litt A., Kramer E.M. The ABC model and the diversification of floral organ identity. Semin. Cell Dev. Biol. 2010;21:129–137. doi: 10.1016/j.semcdb.2009.11.019. [DOI] [PubMed] [Google Scholar]
  • 17.Becker A., Theissen G. The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Mol. Phylogenet. Evol. 2003;29:464–489. doi: 10.1016/S1055-7903(03)00207-0. [DOI] [PubMed] [Google Scholar]
  • 18.Lu J.J., Luo M.F., Wang L., Li K.P., Yu Y.Y., Yang W.F., Gong P.C., Gao H.H. The Physalis floridana genome provides insights into the biochemical and morphological evolution of Physalis fruits. Hortic. Res. 2021;8:244. doi: 10.1038/s41438-021-00705-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Zhao J., Tian Y., Zhang J.S. The euAP1 protein MPF3 represses MPF2 to specify floral calyx identity and displays crucial roles in Chinese lantern development in Physalis. Plant Cell. 2013;25:2002–2021. doi: 10.1105/tpc.113.111757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.He C.Y., Sommer H., Grosardt B., Huijser P., Saedler H. PFMAGO, a MAGO NASHI-like factor, interacts with the MADS-domain protein MPF2 from Physalis floridana. Mol. Biol. Evol. 2007;24:1229–1241. doi: 10.1093/molbev/msm041. [DOI] [PubMed] [Google Scholar]
  • 21.Ampomah-Dwamena C., Morris B.A., Sutherland P., Veit B., Yao J.L. Down-regulation of TM29, a tomatoSEPALLATA homolog, causes parthenocarpic fruit development and floral reversion. Plant Physiol. 2002;130:605–617. doi: 10.1104/pp.005223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Yu X.H., Chen G.P., Guo X.H., Lu Y., Zhang J.L., Hu J.T., Tian S.B., Hu Z.L. Silencing SlAGL6, a tomato AGAMOUS-LIKE6 lineage gene, generates fused sepal and green petal. Plant Cell Rep. 2017;36:959–969. doi: 10.1007/s00299-017-2129-9. [DOI] [PubMed] [Google Scholar]
  • 23.Leseberg C.H., Eissler C.L., Wang X., Johns M.A., Duvall M.R., Mao L. Interaction study of MADS-domain proteins in tomato. J. Exp. Bot. 2008;59:2253–2265. doi: 10.1093/jxb/ern094. [DOI] [PubMed] [Google Scholar]
  • 24.Olmstead R.G., Bohs L., Migid H.A., Santiago-Valentin E., Garcia V.F., Collier S.M. A molecular phylogeny of the Solanaceae. Source. 2008;57:1159–1181. doi: 10.1002/tax.574010. [DOI] [Google Scholar]
  • 25.Särkinen T., Bohs L., Olmstead R.G., Knapp S. A phylogenetic framework for evolutionary study of the nightshades (Solanaceae): A dated 1000-tip tree. BMC Evol. Biol. 2013;13:214. doi: 10.1186/1471-2148-13-214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Cosgrove D.J. Growth of the plant cell wall. Nat. Rev. Mol. Cell Biol. 2005;6:850–861. doi: 10.1038/nrm1746. [DOI] [PubMed] [Google Scholar]
  • 27.Cosgrove D.J. Diffuse Growth of Plant Cell Walls. Plant Physiol. 2018;176:16–27. doi: 10.1104/pp.17.01541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Serna L., Martin C. Trichomes: Different regulatory networks lead to convergent structures. Trends Plant Sci. 2006;11:274–280. doi: 10.1016/j.tplants.2006.04.008. [DOI] [PubMed] [Google Scholar]
  • 29.Pattanaik S., Patra B., Singh S.K., Yuan L. An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis. Front. Plant Sci. 2014;5:259. doi: 10.3389/fpls.2014.00259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Qu H.O., Rylosona J.S., Zhao L.X. Cytological observation of Solanum pimpinellifolium L. Microspore Development. Pak. J. Bot. 2015;47:1459–1465. [Google Scholar]
  • 31.Giménez E., Pineda B., Capel J., Antón M.T., Atarés A., Pérez-Martín F., García-Sogo B., Angosto T., Moreno V., Lozano R. Functional analysis of the Arlequin mutant corroborates the essential role of the Arlequin/TAGL1 gene during reproductive development of tomato. PLoS ONE. 2010;5:e14427. doi: 10.1371/journal.pone.0014427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Theißen G., Melzer R., Rümpler F. MADS-domain transcription factors and the floral quartet model of flower development: Linking plant development and evolution. Development. 2016;143:3259–3271. doi: 10.1242/dev.134080. [DOI] [PubMed] [Google Scholar]
  • 33.Smaczniak C., Immink R.G.H., Muiño J.M., Blanvillain R., Busscher M., Busscher-Lange J., Dinh Q.D., Liu S., Westphal A.H., Boeren S., et al. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proc. Natl. Acad. Sci. USA. 2012;109:1560–1565. doi: 10.1073/pnas.1112871109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Dreni L., Zhang D. Flower development: The evolutionary history and functions of the AGL6 subfamily MADS-box genes. J. Exp. Bot. 2016;67:1625–1638. doi: 10.1093/jxb/erw046. [DOI] [PubMed] [Google Scholar]
  • 35.Doyle J.J., Doyle J.L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 1987;19:11–15. [Google Scholar]
  • 36.Feng S.G., Jiang M.Y., Shi Y.J., Jiao K.L., Shen C.J., Lu J.J., Ying Q.C., Wang H.Z. Application of the ribosomal DNA ITS2 region of Physalis (Solanaceae): DNA barcoding and phylogenetic study. Front. Plant Sci. 2016;7:1047. doi: 10.3389/fpls.2016.01047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Peralta I.E., Spooner D.M. Granule-bound starch synthase (GBSSI) gene phylogeny of wild tomatoes (Solanum L. section Lycopersicon) Am. J. Bot. 2001;88:1888–1902. doi: 10.2307/3558365. [DOI] [PubMed] [Google Scholar]
  • 38.Kumar S., Stecher G., Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Singh R.J. Plant Cytogenetics. 3rd ed. CRC Press; Boca Raton, FL, USA: 2017. [Google Scholar]
  • 40.Ruzin S.E. Plant Microtechnique and Microscopy. Oxford University Press; New York, NY, USA: 1999. [Google Scholar]
  • 41.O’Brien T.P., Feder N., McCully M.E. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma. 1964;59:368–373. doi: 10.1007/bf01248568. [DOI] [Google Scholar]
  • 42.Bozzola J.J., Russell L.D. Electron Microscopy: Principles and Techniques for Biologists. 2nd ed. Jones and Bartlett; Sudbury, MA, USA: 1999. [Google Scholar]
  • 43.Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M.W., Shipley G.L. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 2009;55:611–622. doi: 10.1373/clinchem.2008.112797. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

plants-15-02524-s001.zip (372.6KB, zip)

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Articles from Plants are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES