Expression In Pistils Research Articles

Expression patterns of ABCE model genes during flower development of melon (Cucumis melo L.)

In production, most cultivars of melon are andromonoecious and characterized by carrying both male and bisexual flowers on the same plant. In this study, four A-class genes (CmAP1a, CmAP1b, CmAP2a and CmAP2b), two B-class genes (CmAP3 and CmPI), two C-class genes (CmAGa and CmAGb) and four E-class genes (CmSEP1,2,3,4) were identified in melon. However, no D-class gene of melon was identified. The conserved domains of ABCE function proteins showed relatively high similarity between Arabidopsis and melon. The expression patterns of ABCE homeotic genes in different flower buds of melon suggested that transcripts of CmAP1a, CmPI and CmSEP1 in bisexual buds were significantly lower than that in male flower buds, while the expression levels of CmAGa, CmAGb and CmSEP4 in bisexual flower buds were significantly higher than that in male flower buds. There was no significant difference in expression levels of other ABCE model genes between male buds and bisexual buds. Subsequently, qRT-PCR was performed in different floral organs of bisexual flowers in melon. For A class genes, CmAP1a and CmAP1b showed the highest accumulation in sepals than petals, stamens and pistil, while CmAP2a and CmAP2b revealed the highest expression in pistil than other three floral organs. For B class genes, CmAP3 and CmPI were highly accumulated in petals and stamens though CmAP3 also showed abundant accumulation in pistil. For C class genes, the expression levels of CmAGa and CmAGb were higher in stamens and pistil than that in sepals and petals. For E class genes, CmSEP1 showed higher expression level in sepals and petals than stamens and pistil. CmSEP2, CmSEP3 and CmSEP4 showed the highest accumulation in pistil than other floral organs. These results provided a theoretical basis for studying the function of ABCE homeotic genes in floral organs development of melon.

Molecular Characterization and Expression Patterns of the HkSVP Gene Reveal Distinct Roles in Inflorescence Structure and Floral Organ Development in Hemerocallis fulva.

SHORT VEGETATIVE PHASE (SVP) genes are members of the well-known MADS-box gene family that play a key role in regulating vital developmental processes in plants. Hemerocallis are perennial herbs that exhibit continuous flowering development and have been extensively used in landscaping. However, there are few reports on the regulatory mechanism of flowering in Hemerocallis. To better understand the molecular basis of floral formation of Hemerocallis, we identified and characterized the SVP-like gene HkSVP from the Hemerocallis cultivar ‘Kanai Sensei’. Quantitative RT-PCR (qRT-PCR) indicated that HkSVP transcript was mainly expressed in the vegetative growth stage and had the highest expression in leaves, low expression in petals, pedicels and fruits, and no expression in pistils. The HkSVP encoded protein was localized in the nucleus of Arabidopsis protoplasts and the nucleus of onion epidermal cells. Yeast two hybrid assay revealed that HKSVP interacted with Hemerocallis AP1 and TFL1. Moreover, overexpression of HkSVP in Arabidopsis resulted in delayed flowering and abnormal phenotypes, including enriched trichomes, increased basal inflorescence branches and inhibition of inflorescence formation. These observations suggest that the HkSVP gene may play an important role in maintaining vegetative growth by participating in the construction of inflorescence structure and the development of flower organs.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

Transcriptomic analysis reveals ethylene signal transduction genes involved in pistil development of pumpkin.

Development of female flowers is an important process that directly affects the yield of Cucubits. Little information is available on the sex determination and development of female flowers in pumpkin, a typical monoecious plant. In the present study, we used aborted and normal pistils of pumpkin for RNA-Seq analysis and determined the differentially expressed genes (DEGs) to gain insights into the molecular mechanism underlying pistil development in pumpkin. A total of 3,817 DEGs were identified, among which 1,341 were upregulated and 2,476 were downregulated. The results of transcriptome analysis were confirmed by real-time quantitative RT-PCR. KEGG enrichment analysis showed that the DEGs were significantly enriched in plant hormone signal transduction and phenylpropanoid biosynthesis pathway. Eighty-four DEGs were enriched in the plant hormone signal transduction pathway, which accounted for 12.54% of the significant DEGs, and most of them were annotated as predicted ethylene responsive or insensitive transcription factor genes. Furthermore, the expression levels of four ethylene signal transduction genes in different flower structures (female calyx, pistil, male calyx, stamen, leaf, and ovary) were investigated. The ethyleneresponsive DNA binding factor, ERDBF3, and ethylene responsive transcription factor, ERTF10, showed the highest expression in pistils and the lowest expression in stamens, and their expression levels were 78- and 162-times more than that in stamens, respectively. These results suggest that plant hormone signal transduction genes, especially ethylene signal transduction genes, play an important role in the development of pistils in pumpkin. Our study provides a theoretical basis for further understanding of the mechanism of regulation of ethylene signal transduction genes in pistil development and sex determination in pumpkin.

Influence of drought stress on pistil physiology and reproductive success of two Gossypium hirsutum cultivars differing in drought tolerance.

The causes of reproductive failure under drought stress (DS) are poorly understood. We hypothesized that reproductive failure was related to drought-induced changes in pistil biochemistry. To address this hypothesis, a water deficit-induced experiment was conducted with two cotton cultivars (Dexiamian 1, drought tolerant; Yuzaomian 9110, drought sensitive). Results showed that DS decreased the photosynthesis of subtending leaf and downregulated sucrose transporter gene (GhSUT-1) expression in pistil for both cultivars, resulting in lower pistil carbon accumulation which was reflected in the decreased starch accumulation. Lower starch, as potential energy, and adenosine triphosphate (ATP), as direct energy, in droughted pistils suggested less energy for pollen tube entrance into ovules, reducing the fertilized ovule number and fertilization efficiency. Further, although pistil peroxidase activity increased under DS, a higher hydrogen peroxide (H2 O2 ) level still was measured in droughted pistils than well-watered pistils, damaging reproductive activities. Moreover, larger decreases in photosynthesis, pistil GhSUT-1 expression, carbon accumulation, starch and ATP contents caused by DS for Yuzaomian 9110 than Dexiamian 1, and different responses of superoxide dismutase and catalase activities, and ascorbic acid and H2 O2 contents to DS between the two cultivars might be the reasons causing a greater decrease in fertilization efficiency for Yuzaomian 9110 than Dexiamian 1 under DS. Thus, we suggest that decreased ovule fertilization under DS was related to the disorganized carbohydrate metabolism and inefficient antioxidant defense in droughted pistils, and the effects of DS on pistil carbohydrate metabolism and antioxidant defense were more significant for drought-sensitive cultivars than drought-tolerant cultivars.

ITRAQ-based quantitative proteomic analysis of pistils and anthers from self-incompatible and self-compatible almonds with the S f haplotype

Almond [Prunus dulcis (Miller) D.A. Webb] exhibits self-incompatibility of the gametophytic type, which is controlled by a locus named S with expression in pistils (S-RNase) and in pollen (SFB protein). Recent studies support the involvement of other unidentified components (named modifier factors) in the incompatibility system of this species. A few proteomic studies have tried to identify modifier factors in Prunus species using comparative proteomics of pollinated pistils. However, there are no studies in which the pollen and un-pollinated pistils from self-incompatible and self-compatible Prunus species are compared. In the present work, IEF and RT-PCR analyses of pistils from two almond selections with the S f haplotype but different incompatibility phenotypes revealed that lack of ribonuclease activity in the self-compatible individual is not due to the absence of Sf-RNase transcripts. To identify the proteins differentially expressed in the pistils and anthers of these selections, which could be candidates for modifier factors, isobaric tags for relative and absolute quantitation and 2D-nano-liquid chromatography–electrospray ionisation tandem mass spectrometry were carried out. After a BLAST search against the UniProtKB/SwissProt database, 23 proteins were identified as differentially expressed in pistils and 17 proteins were identified as differentially expressed in anthers. Grouping these proteins into functional categories revealed that most of the pistil proteins had a metabolic function, followed by proteins with a stress resistance and defence function. In the case of anthers, metabolic proteins together with stress resistance and defence proteins were also predominant, although this time in equal proportion. Moreover, the differentially expressed proteins were associated with particular genes, which were located in the Prunus persica Whole Genome version 1.0. Some of the proteins identified here have been linked to pollen development, to pollen dynamics or to pollen–pistil interactions. These results not only provide proteomic profiles of differential expression in mature pistils and anthers, but could also serve as a reference for other comparative proteomic studies of pollinated pistils in almond and in other species with the same incompatibility system.

Analysis of phosphoproteome in rice pistil.

As the female reproductive part of a flower, the pistil consists of the ovary, style, and stigma, and is a critical organ for the process from pollen recognition to fertilization and seed formation. Previous studies on pollen-pistil interaction mainly focused on gene expression changes with comparative transcriptomics or proteomics method. However, studies on protein PTMs are still lacking. Here we report a phosphoproteomic study on mature pistil of rice. Using IMAC enrichment, hydrophilic interaction chromatography fraction and high-accuracy MS instrument (TripleTOF 5600), 2347 of high-confidence (Ascore ≥ 19, p ≤ 0.01), phosphorylation sites corresponding to 1588 phosphoproteins were identified. Among them, 1369 phosphorylation sites within 654 phosphoproteins were newly identified; 41 serine phosphorylation motifs, which belong to three groups: proline-directed, basophilic, and acidic motifs were identified after analysis by motif-X. Two hundred and one genes whose phosphopeptides were identified here showed tissue-specific expression in pistil based on information mining of previous microarray data. All MS data have been deposited in the ProteomeXchange with identifier PXD000923 (http://proteomecentral.proteomexchange.org/dataset/PXD000923). This study will help us to understand pistil development and pollination on the posttranslational level.

Expression Pattern of Class B Gene PAP3 in Flower Development of Pepper

Class B gene APETALA3 (AP3) plays a key role in the development of petals and stamens. Here, we investigated the expression pattern of PAP3 gene (genbank accession number: HM104635) in the buds of cytoplasmic male sterility line 121A and its near-isogenic restorer line 121C at four developmental stages and analyzed the possible association between Class B genes and cytoplasmic male sterility of pepper. Semi-quantitative PCR and quantitative real-time RT-PCR (qRT-PCR) as well as RNA in situ hybridization showed increased expression of PAP3 at late phase of anther development and its higher expression in restorer line compared with sterility line indicating PAP3’s role at late developmental stage of anther and suppressed expression in sterility line. RNA in situ hybridization showed Class B gene features: high abundance in stamen and petal; lower expression in pistil; no expression in sepal. Results of transient expression in onion epidermal cells also showed PAP3 localized in the nucleus, which is consistent with the expression pattern of transcription factors of MADS-box gene family.

Genetic features of a pollen-part mutation suggest an inhibitory role for the Antirrhinum pollen self-incompatibility determinant

Self-incompatibility (SI), an important barrier to inbreeding in flowering plants, is controlled in many species by a single polymorphic S-locus. In the Solanaceae, two tightly linked S-locus genes, S-RNase and SLF (S-locus F-box)/SFB (S-haplotype-specific F-box), control SI expression in pistil and pollen, respectively. The pollen S-determinant appears to function to inhibit all but self S-RNase in the Solanaceae, but its genetic function in the closely-related Plantaginaceae remains equivocal. We have employed transposon mutagenesis in a member of the Plantaginaceae (Antirrhinum) to generate a pollen-part SI-breakdown mutant Pma1 (Pollen-part mutation in Antirrhinum1). Molecular genetic analyses showed that an extra telocentric chromosome containing AhSLF-S ( 1 ) is present in its self-compatible but not in its SI progeny. Furthermore, analysis of the effects of selection revealed positive selection acting on both SLFs and SFBs, but with a stronger purifying selection on SLFs. Taken together, our results suggest an inhibitor role of the pollen S in the Plantaginaceae (as represented by Antirrhinum), similar to that found in the Solanaceae. The implication of these findings is discussed in the context of S-locus evolution in flowering plants.

The identification and characterization of a novel transcription factor LeCBP, a potential regulator of the tomato stylar Chi2;1 promoter

A gene, LeCBP encoding a protein containing a PHD zinc finger motif and a bipartite nuclear localization signal, was identified from tomato pistils by its binding ability to a specific DNA motif using a yeast one-hybrid system. The LeCBP has the ability to bind specifically to a 20 bp region of the promoter of Chi2;1, encoding a pistil chitinase. LeCBP also has the ability to activate the yeast URA3 reporter gene in yeast. An analysis of the temporal and spatial patterns of gene expression through northern blotting and in situ hybridization has demonstrated the highest levels of expression in mature tomato pistils where the mRNA was localized along the transmitting tracks of the style as well as on the ovules and ovary walls. The specific binding property of LeCBP with Chi2;1 promoter may be used to engineer high levels of expression in pistils of transgenic plants.

Heterogeneous expression patterns and separate roles of the SEPALLATA gene LEAFY HULL STERILE1 in grasses.

SEPALLATA (SEP) genes exhibit distinct patterns of expression and function in the grass species rice (Oryza sativa) and maize (Zea mays), suggesting that the role of the genes has changed during the evolution of the family. Here, we examine expression of the SEP-like gene LEAFY HULL STERILE1 (LHS1) in phylogenetically disparate grasses, reconstruct the pattern of gene expression evolution within the family, and then use the expression patterns to test hypotheses of gene function. Our data support a general role for LHS1 in specifying determinacy of the spikelet meristem and also in determining the identity of lemmas and paleas; these two functions are separable, as is the role of the gene in specifying floret meristems. We find no evidence that LHS1 determines flower number; it is strongly expressed in all spikelet meristems even as they are producing flowers, and expression is not correlated with eventual flower number. LHS1 expression in only the upper flowers of the spikelet appears to be the ancestral state; expression in all flowers is derived in subfamily Pooideae. LHS1 expression in pistils, stamens, and lodicules varies among the cereals. We hypothesize that LHS1 may have affected morphological diversification of grass inflorescences by mediating the expression of different floral identity genes in different regions of the floret and spikelet.

CIS-REGULATORY ELEMENTS FOR PISTIL SPECIFIC EXPRESSION IN S-RNASE PROMOTER REGION OF JAPANESE PEAR (PYRUS PYRIFOLIA NAKAI)

The pistil-specific and temporal expression of Japanese pear S-RNase gene is regulated by S-RNase promoter region. To identify the cis-regulatory elements on the S-RNase promoter of Japanese pear, we analysed GUS activities in transgenic tobacco plants carrying 9 sequential deletions of S3-RNase promoter fused to the -glucuronidase (GUS) gene. GUS activity was detected quantitatively in the pistils from the transgenic plants carrying the promoter deleted from -747 to -310. GUS activity was localized in the stigmatic secretory zone, and the transmitting tissues in the pistils but not in other tissues; pollen, anthers, petals, sepals, leaves and roots. GUS activities in the pistils increased steadily from 4 days before flowering. No transgenic pistils with deletions carrying -255 or shorter promoters displayed GUS activity, indicating that the critical cis-regulatory elements for pistil specific expression were present between -310 and -255 of S3-RNase promoter region. Furthermore, deletions from -552 to -356 led to the drastic decrease of GUS activity in the transgenic pistil, which assumes that enhancer-like element for the high level of expression in pistils are present within this region.

An alcohol dehydrogenase-like gene is specifically expressed in potato pistils

No hybridization was detected with mRNA from The reproductive function of the pistil requires the production other flower and vegetative organs. Only after very long of compounds essential for pollen tube growth. A cold-plaque exposure was a faint signal seen in all organs examined. screening of a pollinated pistil cDNA library of Solanum Analysis of the temporal expression pattern of the cp67 gene tuberosum resulted in the isolation of cDNA clone cp67. in whole flower buds indicated that transcripts start to accu- Northern blot analyses revealed that cp67 is specifically mulate in buds of 5-6 mm and were more abundant towards expressed in potato pistils and in a limited number of plant the end of flower development (data not shown). species. The deduced CP67 protein displays similarity to The conservation of cp67 gene expression in pistils of other long-chain zinc-containing alcohol dehydrogenases (ADHs), plant species was investigated. As it can be seen in Fig. 1B, although at a similarity level much lower than between other cp67 transcripts were only detectable in pistils of S. tuberosum plant ADHs. and Datura. No hybridization signals were observed in mRNA of pistils of Nicotiana tabacum, Petunia hybrida and

A promoter directing high level expression in pistils of transgenic plants.

The promoter of the potato (Solanum tuberosum L.) SK2 gene, encoding a pistil-specific basic endochitinase, was cloned. Various fragments of the SK2-promoter, from 1 kb down to 0.23 kb in length, were fused to the GUS reporter gene. Chimaeric SK2 promoter-GUS fusion constructs were transformed into potato by Agrobacterium tumefaciens-mediated transformation. The SK2-GUS transgenic potato plants exhibited a highly specific GUS activity in the pistil. Expression in the pistil was shown to be developmentally regulated. In addition to the GUS activity in pistils, transgenic plants also showed a much weaker ectopic expression in anthers. In other tissues no systematic expression was detectable. All SK2 promoter fragments analysed conferred pistil-specific expression without significant qualitative or quantitative differences, demonstrating that the regulatory elements mediating this expression pattern are located within a 230 bp SK2 promoter fragment. The SK2 promoter may be used to engineer high levels of expression in pistils of transgenic plants.

Molecular Cloning of Members of the <italic>S</italic>-Multigene Family in Self-Incompatible <italic>Brassica campestris</italic> L.

We isolated 12 groups of genomic clones that contained SLG-homologous regions from a genomic library constructed from an S9 homozygote of self-incompatible Brassica campestris. Both SLG9 and SRK9 genomic clones, which are located within the self-incompatibility (S) locus, were included in these groups. The promoter regions of SLG9 and SRK9 were completely identical for at least 200 bp upstream from their respective initiation codons (ATG). The five sequence elements (boxes I to V) that are conserved in the promoters of SLG and SRK genes were also found in the SLG9 and SRK9 clones. However, one conserved element (box III) unexpectedly lacked 7 of 11 bp, although box III has been considered necessary for expression in pistil. The other ten groups of genomic clones were classified into six SRK-like groups and four SLG-like groups. These results indicate that SLG, SRK, SLG-like, and SRK-like genes form a large S-multigene family in B. campestris.

New Perspectives in Pollen Biology and Fertilization

The reproductive biology of flowering plants is one of the most rapidly developing fields in plant science, due largely to the introduction of new molecular and computer technology. Today, the techniques of cell biology have new implications for plant reproductive success. This success can be estimated using measurements of pollen quantity and quality, pollen germination and tube growth, and successful fertilization. The complex array of interrelated processes constituting reproductive success is a focus for plant biologists who need to understand, modify and exploit them for plant improvement. The success of fertilization is reviewed in terms of (1) differential gene expression in pistil and pollen, and (2) effectiveness of the gametes. On the female side, potential genes of interest include those regulating stigma receptivity, self-incompatibility and ovule viability. On the male side, interest centres on molecular analysis of certain pollen-specific genes which affect male transmission at fertilization. Experimental analysis of the effectiveness of the male and female gametes is being approached in two distinct ways: (1) analysis of their nature and organization using image analysis combined with interactive computer graphics, and (2) isolation of the gametes using cell fractionation technology. Examples of these approaches are given for sperm cell biology, where there are two important questions to be answered: (1) what is the organization of the pair of sperm cells and their delivery system within the pollen tube? and (2) is there specific recognition between the sperm cells and the female gametes — the egg and central cell?