[show abstract][hide abstract] ABSTRACT: Although self-incompatibility (SI) in plants has been studied extensively, far less is known about interspecific reproductive barriers. One interspecific barrier, known as unilateral incongruity or incompatibility (UI), occurs when species display unidirectional compatibility in interspecific crosses. In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of S. pennellii using a semi-in vivo system to track pollen-tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 3-4 days before flower opening. The developmental expression dynamics of known SI factors, S-RNases and HT proteins, was also examined. The accumulation of HT-A protein coincided temporally and spatially with UI barriers in developing pistils. Proteomic analysis of stigma/styles from key developmental stages showed a switch in protein profiles from cell-division-associated proteins in immature stigma/styles to a set of proteins in mature stigma/styles that included S-RNases, HT-A protein and proteins associated with cell-wall loosening and defense responses, which could be involved in pollen-pistil interactions. Other prominent proteins in mature stigma/styles were those involved in lipid metabolism, consistent with the accumulation of lipid-rich material during pistil maturation.
Journal of Experimental Botany 11/2012; · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: In Solanaceae, S-RNase and SLF/SFB (S-locus F-box) interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil modifier genes -120K and HT-B- have been identified in Nicotiana. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (Nicotiana alata Stigma Expressed Protein) that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana transgenic plants disrupts S-specific pollen rejection, therefore, NaStEP is a novel stigma modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. From these data, the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.
[show abstract][hide abstract] ABSTRACT: The purpose of this investigation was to explore students’ epistemic beliefs and conceptual understanding of biotechnology.
Epistemic beliefs can influence reasoning, how individuals evaluate information, and informed decision making abilities. These
skills are important for an informed citizenry that will participate in debates regarding areas in science such as biotechnology.
We report on an in-depth case study analysis of three undergraduate, non-science majors in a biotechnology course designed
for non-biochemistry majors. We selected participants who performed above average and below average on the first in-class
exam. Data from multiple sources—interviews, exams, and a concept instrument—were used to construct (a) individual profiles
and (b) a cross-case analysis of our participants’ conceptual development and epistemic beliefs from two different theoretical
perspectives—Women’s Ways of Knowing and the Reflective Judgment Model. Two independent trained researchers coded all case
records independently for both theoretical perspectives, with resultant initial Cohen’s kappa values above .715 (substantial
agreement), and then reached consensus on the codes. Results indicate that a student with more sophisticated epistemology
demonstrated greater conceptual understandings at the end of the course than a student with less sophisticated epistemology,
even though the latter performed higher initially. Also a student with a less sophisticated epistemology and low initial conceptual
performance does not demonstrate gains in their overall conceptual understanding. Results suggest the need for instructional
interventions fostering epistemological development of learners in order to facilitate their conceptual growth.
KeywordsEpistemology–Case study–Biotechnology education–Qualitative research–Science education–University–Undergraduate
Research in Science Education 04/2012; 42(2):353-371. · 1.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse,
but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships
with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and
interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively
correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen
tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species×short-pistil species
crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil
species×long-pistil species. Taken together, these barriers can generally be described by a ‘pistil-length mismatch’ rule;
in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute
to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths.
Sexual Plant Reproduction 04/2012; 21(3):183-195. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Expressing a pollen self-incompatibility gene from Papaver rhoeas (poppy) in Arabidopsis thaliana renders the latter sensitive to an exquisitely precise induced cell death response. This simple system may have wide application in biotechnology and research.
Current biology: CB 02/2012; 22(3):R86-7. · 10.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: Self-incompatibility (SI) is a common form of genetically-controlled mate-selection that prevents mating between closely related plants of the same species. SI occurs in about half of all flowering plant species. It has been studied extensively in the Papaveraceae (poppy), Brassicaceae (Arabidopsis, cabbage etc), Solanaceae (potato, tomato etc), Plantaginaceae (snapdragon) and Rosaceae (apple, cherry and peach etc). The self-recognition inherent in self-incompatibility has similarities with animal and plant immunity systems giving rise to speculation that the systems are related. Both systems display balancing selection, 'self/nonself' recognition, high polymorphism, high specificity and there are also some similarities in the rejection mechanisms deployed in the two systems. Whether these systems have diverged from a common predecessor is discussed, however similarities may be driven more by biological problems and the available molecular machinery to solve them than by an evolutionary relationship.
Advances in experimental medicine and biology 01/2012; 738:108-23. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background: S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity. SCOPE: The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility. CONCLUSIONS: Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI.
Annals of Botany 07/2011; 108(4):647-58. · 3.45 Impact Factor
[show abstract][hide abstract] ABSTRACT: Wild tomato species in Solanum Section Lycopersicon often exhibit two types of reproductive barriers: self-incompatibility (SI) and unilateral incompatibility or incongruity (UI), wherein the success of an inter-specific cross depends on the direction of the cross. UI pollen rejection often follows the 'SI × SC' rule, i.e. pistils of SI species reject the pollen of SC (self-compatible) species but not vice versa, suggesting that the SI and UI pollen rejection mechanisms may overlap. In order to address this question, pollen tube growth was measured after inter-specific crosses using wild tomato species as the female parents and pollen from cultivated tomato (Solanum lycopersicum). Two modes of UI pollen rejection, early and late, were observed, and both differed from SI pollen rejection. The structure and expression of known stylar SI genes were evaluated. We found that S-RNase expression is not required for either the early or late mode of UI pollen rejection. However, two HT family genes, HT-A and HT-B, map to a UI QTL. Surprisingly, we found that a gene previously implicated in SI, HT-B, is mutated in both SI and SC S. habrochaites accessions, and no HT-B protein could be detected. HT-A genes were detected and expressed in all species examined, and may therefore function in both SI and UI. We conclude that there are significant differences between SI and UI in the tomato clade, in that pollen tube growth differs between these two rejection systems, and some stylar SI factors, including S-RNase and HT-B, are not required for UI.
The Plant Journal 11/2010; 64(3):367-78. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The tomato clade within the genus Solanum has numerous advantages for mechanistic studies of reproductive isolation. Its thirteen closely related species, along with four closely allied Solanum species, provide a defined group with diverse mating systems that display complex interspecific reproductive barriers. Several kinds of pre- and postzygotic barriers have already been identified within this clade. Well-developed genetic maps, introgression lines, interspecific bridging lines, and the newly available draft genome sequence of the domesticated tomato (Solanum lycopersicum) are valuable tools for the genetic analysis of interspecific reproductive barriers. The excellent chromosome morphology of these diploid species allows detailed cytological analysis of interspecific hybrids. Transgenic methodologies, well developed in the Solanaceae, allow the functional testing of candidate reproductive barrier genes as well as live imaging of pollen rejection events through the use of fluorescently tagged proteins. Proteomic and transcriptomics approaches are also providing new insights into the molecular nature of interspecific barriers. Recent progress toward understanding reproductive isolation mechanisms using these molecular and genetic tools is assessed in this review.
Sexual Plant Reproduction 11/2010; 24(3):171-87. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: The endomembrane system offers many potential points where plant mating can be effectively controlled. This results from two basic features of angiosperm reproduction: the requirement for pollen tubes to pass through sporophytic tissues to gain access to ovules and the physiology of pollen tube growth that provides it with the capacity to do so. Rapid pollen tube growth requires extravagant exocytosis and endocytosis activity as cell wall material is deposited and membrane is recovered from the actively growing tip. Moreover, recent results show that pollen tubes take up a great deal of material from the pistil extracellular matrix. Regarding the stigma and style as organs specialized for mate selection focuses attention on their complementary roles in secreting material to support the growth of compatible pollen tubes and discourage the growth of undesirable pollen. Since these processes also involve regulated activities of the endomembrane system, the potential for regulating mating by controlling endomembrane events exists in both pollen and pistil.
Journal of Experimental Botany 04/2010; 61(7):2001-13. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Charles Darwin made extensive observations of the pollination biology of a wide variety of plants. He carefully documented the consequences of self-pollination and described species that were self-sterile but that could easily be crossed with other plants of the same species. He believed that compatibility was controlled by the 'mutual action' of pollen and pistil contents. A genetic model for self-sterility was developed in the early 1900 s based on studies of the compatibility relationships among, what are now referred to as, self-incompatible (SI) Nicotiana species. Today, it is believed that SI in these species is controlled by an interaction between S-RNases produced in the pistil and F-box proteins expressed in pollen and, moreover, that this S-RNase-based SI system is shared by a great diversity of other plant species. Current research is aimed at understanding how the mutual actions of these S-gene products function in the physiological context of pollen tube growth.
Journal of Experimental Botany 02/2009; 60(4):1069-81. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: As pollen tubes grow toward the ovary, they are in constant contact with the pistil extracellular matrix (ECM). ECM components are taken up during growth, and some pistil molecules exert their effect inside the pollen tube. For instance, the Nicotiana alata 120-kD glycoprotein (120K) is an abundant arabinogalactan protein that is taken up from the ECM; it has been detected in association with pollen tube vacuoles, but the transport pathway between these compartments is unknown. We recently identified a pollen C2 domain-containing protein (NaPCCP) that binds to the carboxyl-terminal domain of 120K. As C2 domain proteins mediate protein-lipid interactions, NaPCCP could function in intracellular transport of 120K in pollen tubes. Here, we describe binding studies showing that the NaPCCP C2 domain is functional and that binding is specific for phosphatidylinositol 3-phosphate. Subcellular fractionation, immunolocalization, and live imaging results show that NaPCCP is associated with the plasma membrane and internal pollen tube vesicles. Colocalization between an NaPCCPgreen fluorescent protein fusion and internalized FM4-64 suggest an association with the endosomal system. NaPCCP localization is altered in pollen tubes rejected by the self-incompatibility mechanism, but our hypothesis is that it has a general function in the transport of endocytic cargo rather than a specific function in self-incompatibility. NaPCCP represents a bifunctional protein with both phosphatidylinositol 3-phosphate- and arabinogalactan protein-binding domains. Therefore, it could function in the transport of pistil ECM proteins in the pollen tube endomembrane system.
[show abstract][hide abstract] ABSTRACT: Pollen tube growth is influenced by interaction between pollen proteins and the pistil extracellular matrix. The transmitting tract-specific glycoprotein (NaTTS) and 120-kDa glycoprotein (120K) are two pistil arabinogalactan proteins (AGPs) that share a conserved C-terminal domain (CTD) and directly influence pollen tubes in Nicotiana alata. 120K and other extracellular matrix proteins are taken up and transported to vacuoles of growing pollen tubes. We hypothesize that signaling and trafficking processes inside pollen tubes are important for controlling pollen tube growth. We performed a yeast two-hybrid screen of pollen cDNAs using sequences from 120K and NaTTS as baits. We found that an S-RNase-binding protein (SBP1), a C2 domain-containing protein (NaPCCP), and a putative cysteine protease bound to the AGP baits. SBP1 from Petunia hybrida and Solanum chacoense is a putative E3 ubiquitin ligase that binds to S-RNase and other proteins. C2 domain-containing proteins bind lipids and can regulate myriad cellular processes. Cysteine proteases are often associated with the degradation of vacuolar proteins. Expression analysis revealed that transcripts for these proteins are expressed in mature pollen. NaPCCP and NaSBP1 were characterized further because of their potential roles in signaling and trafficking. In vitro pull-down assays verified binding between maltose-binding protein (MBP) fusions, MBP::NaPCCP or MBP::NaSBP1 and glutathione S-transferase (GST), GST::AGP CTD fusions. NaSBP1 binds to the AGP CTDs through its helical and RING domains. NaPCCP binds through its C-terminal region. Binding between NaPCCP and NaSBP1 and the pistil AGPs may contribute to signaling and trafficking inside pollen tubes growing in planta.
Journal of Biological Chemistry 09/2008; 283(40):26965-73. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: HT-family proteins have been identified in Nicotiana, Solanum, and Petunia. HT-B-type proteins are implicated in S-RNase-based self-incompatibility, but the functions of other family members are unknown. Screening for cDNA sequences with an expression pattern similar to HT-B in Nicotiana alata revealed a new group of small HT-family proteins, designated HT-M. HT-M proteins resemble HT-B in several respects: their pistil-specific expression pattern is indistinguishable from HT-B, they pellet with a microsome fraction, and their abundance decreases after pollination. Unlike HT-B, there is no S-specificity to this response, and RNAi experiments show that HT-M proteins are not necessary for self-incompatibility. Identification of a third group of pistil-specific HT-family proteins helps better define the characteristics of the family and allowed identification of putative new family members. By searching the databases with only the most conserved HT-family sequence elements, the signal sequence and cysteine motifs, we identified nodulin-24-like proteins and several small glycine-rich proteins as putative HT-family members. Like HT-M and HT-B, nodulin-24 is membrane associated. We propose that the conserved features in HT-family proteins are important for targeting or modification and refer to the broader family that includes both HT- and nodulin-24-like proteins as the HT/NOD-24-family.
[show abstract][hide abstract] ABSTRACT: After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen-stigma interactions that regulate pollen tube growth in Nicotiana.
Journal of Experimental Botany 02/2008; 59(11):3187-201. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the process of pollination, haploid pollen germinates on the stigma surface and a pollen tube grows through the diploid tissues of the pistil toward the ovary. The pistil has two basic functions: to prevent unwanted pollen from gaining access to the ovary and to support the growth of desirable pollen. Pollen-pistil signaling allows these different types of pollen to be distinguished. Self-incompatibility (SI) systems, controlled by the S locus, are the best-understood pollen-pistil signaling systems. Other SI systems have been investigated at the molecular level, but the physiology of pollen tube rejection is best understood in the field poppy, Papaver rhoeas. This species has a gametophytic SI system: Pollen is rejected when its S haplotype is the same as either of the two S haplotypes expressed in the diploid pistil. Recent advances reveal new ways that SI controls pollen tube metabolism. A soluble pyrophosphatase is down-regulated as part of the rapid SI response, and, over the long term, perturbations of the actin cytoskeleton lead to programmed cell death in incompatible pollen tubes. Manipulating incompatible pollen tube metabolism in this way may leave more resources available for supporting the growth of compatible pollen tubes, the complementary function of the pistil.
[show abstract][hide abstract] ABSTRACT: S-RNase-based self-incompatibility (SI) is the most widespread form of genetically controlled mate selection in plants. S-RNase controls pollination specificity in the pistil, while the newly discovered SLF/SFB controls pollination specificity in the pollen. A widely discussed model suggests that compatibility is explained by ubiquitylation and degradation of nonself-S-RNase and that, conversely, incompatibility is caused by failure to degrade self-S-RNase. This model is consistent with the long-standing view that S-RNase inhibition is central to SI. Recent results show, however, that S-RNase is compartmentalized in pollen tubes and, significantly, that compatibility might not require SLF/SFB. S-RNase compartmentalization and dislocation into the pollen tube cytoplasm might be similar to the trafficking of other cytotoxins such as ricin.
Current Opinion in Plant Biology 01/2007; 9(6):639-46. · 8.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: Self-incompatibility (SI) prevents the production of "self" seed and inbreeding by providing a recognition and rejection system for "self," or genetically identical, pollen. Studies of gametophytic SI (GSI) species at a molecular level have identified two completely different S-genes and SI mechanisms. One GSI mechanism, which is found in the Solanaceae, Rosaceae and Scrophulariaceae, has S-RNase as the pistil S-component and an F-box protein as the pollen S-component. However, non-S-locus factors are also required. In an incompatible situation, the S-RNases degrade pollen RNA, thereby preventing pollen tube growth. Here, in the light of recent evidence, we examine alternative models for how compatible pollen escapes this cytotoxic activity. The other GSI mechanism, so far found only in the Papaveraceae, has a small secreted peptide, the S-protein, as its pistil S-component. The pollen S-component remains elusive, but it is thought to be a transmembrane receptor, as interaction of the S-protein with incompatible pollen triggers a signaling network, resulting in rapid actin depolymerization and pollen tube inhibition and programmed cell death (PCD). Here, we present an overview of what is currently known about the mechanisms involved in regulating pollen tube inhibition in these two GSI systems.