Leaf Protoplasts Research Articles

Low-temperature sensing in olive tree: calcium signalling and cold acclimation

Olive tree is a warm-temperature evergreen tree with low tolerance to frost, although cultivars which differ in terms of cold acclimation have been empirically selected. In herbaceous species, free cytosolic calcium ([Ca 2+] c) is involved in cold acclimation. The objective of this study was to measure [Ca 2+] c signalling in the olive tree during cold acclimation and to assess the possibility of using [Ca 2+] c as an early genotype-selection marker of cold susceptibility. To this end, non-cold-acclimated and cold-acclimated leaf protoplasts of cultivars differing in terms of cold susceptibility were analysed. Cold shocks of various amplitude applied to non-cold-acclimated protoplasts caused consistent and transient increases in [Ca 2+] c. A decrease of 0.05 °C/s (i.e. Δ T/d t=2.5 °C/50 s) was the threshold cooling rate at which a significant increase in [Ca 2+] c could still be observed. When the protoplasts were incubated with either 8-( N, N-di-methylamino)octyl 3,4,5-trimethoxy-benzoate (TMB-8; organelle Ca 2+ channel blocker) or Gd 3+ (plasma membrane Ca 2+ channel blocker), applying the threshold cooling rate, the increase in [Ca 2+] c was partially inhibited, suggesting that both an intracellular release of Ca 2+ and an influx through the plasma membrane are involved. When applying repeated cold shocks, the transient increases in [Ca 2+] c were reduced only when using a non-severe Δ T/d t. In protoplasts subjected to standard acclimation, the increases in [Ca 2+] c were further reduced, or inhibited, depending on the cold susceptibility of the cultivar, suggesting that the Ca 2+ response is involved in a long-term adaptation to cold.

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases by reduction levels of pyridine nucleotides in mitochondria and cytosol of pea leaves

Regulation of NAD- and NADP-dependent isocitrate dehydrogenases (NAD-ICDH, EC 1.1.1.41, and NADP-ICDH, EC 1.1.1.42) by the level of reduced and oxidized pyridine nucleotides has been investigated in pea ( Pisum sativum L.) leaves. The affinities of mitochondrial and cytosolic ICDH enzymes to substrates and inhibitors were determined on partially purified preparations in forward and reverse directions. From the kinetic data, it follows that NADP +- and NAD +-dependent isocitrate dehydrogenases in mitochondria represent a system strongly responding to the intramitochondrial NADPH and NADH levels. The NADPH, NADP +, NADH and NAD + concentrations were determined by subcellular fractionation of pea leaf protoplasts using membrane filtration in mitochondria and cytosol in darkness and in the light under saturating and limiting CO 2 conditions. The cytosolic NADPH/NADP ratio was about 1 and almost constant both in darkness and in the light. In mitochondria, the NADPH/NADP ratio was low in darkness (0.2) and increased in the light, reaching 3 in limiting CO 2 conditions compared to 1 in saturating CO 2. At high reduction levels of NADP and NAD observed at limiting CO 2 in the light, i.e. when photorespiratory glycine is the main mitochondrial substrate, isocitrate oxidation in mitochondria will be suppressed and citrate will be transported to the cytosol (‘citrate valve’), where the cytosolic NADP-ICDH supplies 2-oxoglutarate for the photorespiratory ammonia refixation.

Comparison of hCMV immediate early and CaMV 35S promoters in both plant and human cells

Cauliflower mosaic virus 35S promoter, widely used in transgenic crop plants, is known to be recognized in widely differing kinds of cells. Its activity in human cells may have impact on the risk assessment for the environmental release of genetically modified plants. In this study, transient expression of several constructs containing β-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells. The results showed very low but measurable activity of 35S promoter in human 293T-cells (0.01% of that revealed when using pCMV) and in 293 cells that do not produce SV40 T antigen this activity was even lower. On the other hand, in potato protoplasts, pCMV displayed nearly 1% activity seen with p35S.

Phytoalexin production in grapevine protoplasts during isolation and culture

The production of stilbene phytoalexins was analyzed in Vitis sp. protoplasts during isolation and culture, by HPLC. The results showed that trans-resveratrol could be detected as early as the fourth hour of digestion and accumulated during the digestion process, while the healthy donor material was devoid. Resveratrol production was due to de novo activation of the Vst1 gene encoding for the stilbene synthase (EC 2.3.1.95) responsible for resveratrol formation. When in culture, leaf protoplasts, whatever the genotype, continued to produce large amounts of resveratrol within the first week of culture, whereas, during the second week, a decrease in resveratrol content was observed, in parallel to a decrease in protoplast viability. Other phytoalexins derived from the resveratrol were produced during the culture, mainly an inducible resveratrol dehydrodimer analogous to ε-viniferin and, to a lesser extent, pterostilbene. In order to determine the inducer of phytoalexin production, effects of culture conditions were evaluated on resveratrol production and discussed.

Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses.

Many abiotic stress-inducible genes contain two cis-acting elements, namely a dehydration-responsive element (DRE; TACCGACAT) and an ABA-responsive element (ABRE; ACGTGG/TC), in their promoter regions. We precisely analyzed the 120 bp promoter region (-174 to -55) of the Arabidopsis rd29A gene whose expression is induced by dehydration, high-salinity, low-temperature, and abscisic acid (ABA) treatments and whose 120 bp promoter region contains the DRE, DRE/CRT-core motif (A/GCCGAC), and ABRE sequences. Deletion and base substitution analyses of this region showed that the DRE-core motif functions as DRE and that the DRE/DRE-core motif could be a coupling element of ABRE. Gel mobility shift assays revealed that DRE-binding proteins (DREB1s/CBFs and DREB2s) bind to both DRE and the DRE-core motif and that ABRE-binding proteins (AREBs/ABFs) bind to ABRE in the 120 bp promoter region. In addition, transactivation experiments using Arabidopsis leaf protoplasts showed that DREBs and AREBs cumulatively transactivate the expression of a GUS reporter gene fused to the 120 bp promoter region of rd29A. These results indicate that DRE and ABRE are interdependent in the ABA-responsive expression of the rd29A gene in response to ABA in Arabidopsis.

Development of novel elongated fiber-structure in protoplast cultures of Betula platyphylia and Larix leptolepis

Novel elongated fiber-structures were repeatedly found both in leaf protoplast culture of two clones of Betula platyphylla and in protoplast culture of embryogenic cells of Larix leptolepis. Suboptimum culture conditions for cell division appeared to lead to fiber formation when using multi-well plate culture with varying medium compositions The suboptimum conditions for cell divisions were brought about by (1) plant growth regulators: auxins and cytokinins; (2) pH: 3.5, 4.5, 5.8; (3) divalent cations: CaCl2 and MgCl2; and (4) sugars: sucrose and mannitol. Divalent cations had the most profound effect on fiber formation. Calcium ions were preferred by Betula and magnesium ions were preferred by Larix. Single fiberpurification and micro-staining methods using a micromanipulator were developed. The fibers fluoresced when stained with Calcofluor White and Aniline Blue, which suggested that they were composed of cell wall component(s), including callose (β-1,3-glucan). Electron microscopy showed that fiber bundles of Larix fibers had helical substructures.

Various ion channels in leaf protoplasts from the moss Physcomitrella patens

Many molecular and genetic tools are available for the moss Physcomitrella patens and make this anatomically simple plant an attractive model system. Here, the ionic conductances in the plasma membrane of protoplasts derived from the gametophytic leaves of P. patens were studied using the patch clamp technique. Though the leaves contain only a single layer of uniform appearing cells, the results show some diversity in the current types among protoplasts. In whole cell preparations, both outwardly and inwardly rectifying potassium currents were observed. The currents were time- and voltage-dependent and resemble the currents found in higher plants that are mediated by potassium channels. Current–voltage plots of the tail currents indicate that the currents were mainly carried by K +. The inward potassium channels were highly permeable to ammonium, but not sodium. In standard conditions, using potassium, calcium and chloride as major ions, some cells displayed only outwardly rectifying potassium currents. In addition, time-independent and novel deactivating cation currents were observed in another set of cells. In summary, moss protoplasts display diverse current types, many of which are also observed to be typical in higher plant species.

Increased CO2 uncouples growth from isoprene emission in an agriforest ecosystem.

The emission of isoprene from the leaves of forest trees is a fundamental component of biosphere-atmosphere interactions, controlling many aspects of photochemistry in the lower atmosphere. As almost all commercial agriforest species emit high levels of isoprene, proliferation of agriforest plantations has significant potential to increase regional ozone pollution and enhance the lifetime of methane, an important determinant of global climate. Here we show that growth of an intact Populus deltoides plantation under increased CO2 (800 micromol x mol(-1) and 1,200 micromol x mol(-1)) reduced ecosystem isoprene production by 21% and 41%, while above-ground biomass accumulation was enhanced by 60% and 82%, respectively. Exposure to increased CO2 significantly reduced the cellular content of dimethylallyl diphosphate, the substrate for isoprene synthesis, in both leaves and leaf protoplasts. We identify intracellular metabolic competition for phosphoenolpyruvate as a possible control point in explaining the suppression of isoprene emission under increased CO2. Our results highlight the potential for uncoupling isoprene emission from biomass accumulation in an agriforest species, and show that negative air-quality effects of proliferating agriforests may be offset by increases in CO2.

Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling.

In Arabidopsis, the induction of a dehydration-responsive gene, rd22, is mediated by abscisic acid (ABA). We reported previously that MYC and MYB recognition sites in the rd22 promoter region function as cis-acting elements in the drought- and ABA-induced gene expression of rd22. bHLH- and MYB-related transcription factors, rd22BP1 (renamed AtMYC2) and AtMYB2, interact specifically with the MYC and MYB recognition sites, respectively, in vitro and activate the transcription of the beta-glucuronidase reporter gene driven by the MYC and MYB recognition sites in Arabidopsis leaf protoplasts. Here, we show that transgenic plants overexpressing AtMYC2 and/or AtMYB2 cDNAs have higher sensitivity to ABA. The ABA-induced gene expression of rd22 and AtADH1 was enhanced in these transgenic plants. Microarray analysis of the transgenic plants overexpressing both AtMYC2 and AtMYB2 cDNAs revealed that several ABA-inducible genes also are upregulated in the transgenic plants. By contrast, a Ds insertion mutant of the AtMYC2 gene was less sensitive to ABA and showed significantly decreased ABA-induced gene expression of rd22 and AtADH1. These results indicate that both AtMYC2 and AtMYB2 proteins function as transcriptional activators in ABA-inducible gene expression under drought stress in plants.

Apoptosis-like cell death of Brassica napus leaf protoplasts.

• The cleavage of nuclear DNA into oligonucleosomal fragments that is the hallmark of apoptosis in animal cells occurs during the culture of Brassica napus leaf protoplasts. • The changes in nuclei of cultured Brassica napus leaf protoplasts were studied by propidium iodide (PI) and 4', 6-diamino-2-phenylindole, dihydrochloride (DAPI) staining, transmission electron microscopy, flow cytometry analysis, and DNA laddering staining with ethidium bromide and Southern hybridization. • Free 3'-OH termini of nuclear DNA fragments were labelled with DIG-dUTP, catalyzed by terminal deoxynucleotidyl transferase (TdT), and used as probes for Southern hybridization. This method (TUNEL on membrane) allowed visualization of DNA fragments with 3'-OH termini on a nylon membrane. • These results suggest that loss of viability of protoplast with culture time is accompanied by apoptosis-like cell death. However, the forms or processes undergoing to apoptotic cell death in B. napus leaf protoplasts appears to be different in some details to those in animal cells.

Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors.

Zinc finger transcription factors (TFs(ZF)) were designed and applied to transgene and endogenous gene regulation in stably transformed plants. The target of the TFs(ZF) is the Arabidopsis gene APETALA3 (AP3), which encodes a transcription factor that determines floral organ identity. A zinc finger protein (ZFP) was designed to specifically bind to a region upstream of AP3. AP3 transcription was induced by transformation of leaf protoplasts with a transformation vector that expressed a TF(ZF) consisting of the ZFP fused to the tetrameric repeat of herpes simplex VP16's minimal activation domain. Histochemical staining of beta-glucuronidase (GUS) activity in transgenic AP3GUS reporter plants expressing GUS under control of the AP3 promoter was increased dramatically in petals when the AP3-specific TF(ZF) activator was cointroduced. TF(ZF)-amplified GUS expression signals were also evident in sepal tissues of these double-transgenic plants. Floral phenotype changes indicative of endogenous AP3 factor coactivation were also observed. The same AP3-specific ZFP(AP3) was also fused to a human transcriptional repression domain, the mSIN3 interaction domain, and introduced into either AP3GUS-expressing plants or wild-type Arabidopsis plants. Dramatic repression of endogenous AP3 expression in floral tissue resulted when a constitutive promoter was used to drive the expression of this TF(ZF). These plants were also sterile. When a floral tissue-specific promoter from APETALA1 (AP1) gene was used, floral phenotype changes were also observed, but in contrast the plants were fertile. Our results demonstrate that artificial transcriptional factors based on synthetic zinc finger proteins are capable of stable and specific regulation of endogenous genes through multiple generations in multicellular organisms.

Transient expression of <em>uid</em>A gene in grapevine protoplasts after PEG-mediated transformation

<p style="text-align: justify;">Leaf protoplasts, isolated from <em>Vitis vinifera</em> «Sakasly» and «Muscat d’Alexandrie» and protoplasts from embryogenic tissue of <em>Vitis</em> sp. «Seyval blanc» were incubated in the presence of PEG in a transformation solution containing the plasmid pBI426 which carries the β-glucuronidase (<em>gus</em>) and the neomycin phosphotransferase II (<em>npt</em>II) genes. The treated protoplasts were cultivated in CPW13 medium without kanamycin selection. 48h after the PEG transformation, transient expression of gus gene was detected histochemically and fluorimetrically in the protoplast cultures.</p>

Differential responses of Brassica napus and Petunia hybrida to leaf protoplast isolation stress.

Changes in the response to abiotic stress during the isolation of leaf protoplasts were compared between a recalcitrant species of Brassica napus and regenerating species of Petunia hybrida. Initially, levels of soluble free putrescine (put), spermidine (spd) and spermine (spm) in leaves and protoplasts were determined. The sum of these three polyamines increased in petunia and B. napus leaf protoplasts by 1.6-fold and 1.1-fold, respectively. The soluble free fraction of spd and spm decreased in B. napus but not in petunia protoplasts. During the isolation of leaf protoplasts from B. napus, the ratio of soluble free put to the total PAs almost doubled, but that of spd and spm declined significantly. Petunia leaf protoplasts treated with cyclohexylamine (CHA), an inhibitor of spermidine synthase, accumulated ammonia and soluble putrescine, but lost the soluble spermidine. The soluble polyamine levels of CHA-treated petunia leaf protoplasts corresponded with those in B. napus. Leaves were subjected to abiotic stress during the isolation of protoplasts, namely wounding and osmotic stress which changed soluble free polyamine levels in B. napus and petunia, respectively. Both B. napus and petunia leaf protoplasts showed an increase in ammonia, but total free amino acid content and activation of proteases were only enhanced in B. napus leaf protoplasts. These results suggest that in B. napus wounding initiated senescence of leaf protoplasts during their isolation, leading to a constant production of ethylene early in the culture.

Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth.

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein-NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein-NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.

Endogenous levels of abscisic acid and gibberellins in leaf protoplasts competent for plant regeneration in Betula platyphylla and Populus alba

Differences in endogenous levels of abscisic acid and gibberellinsbetween Betula platyphylla and Populusalba leaf protoplasts were determined using micro-scale extractionand purification steps, including thin layer chromatography ormicro-high-performance-liquid-chromatography and quantification by enzymelinkedimmunosorbent assay or micro-bioassay. The content of abscisic acid was tentimes higher in B. platyphylla than in P.alba on the basis of both cell number and dry weight; in contrast,levels of gibberellins were lower in the former. Leaf protoplasts of bothspecies are competent for plant regeneration through the exogenous supply ofauxins and cytokinins. The function of abscisic acid in these protoplastcultures is discussed in relation to the need for a strong cytokinin,N-(2-chloro-4-pyridyl)-N′-phenylurea (4-CPPU) for colony proliferation inB. platyphylla, in contrast to a weak cytokininrequirementin P. alba.

Temperature-dependent Apoptosis Detected in Hybrids between Nicotiana debneyi and N. tabacum Expressing Lethality

Interspecific hybrids of Nicotiana debneyi × N. tabacum L. show hybrid lethality, which is one of the mechanisms for reproductive isolation. Apoptotic changes were detected in the cells of hybrid seedlings expressing lethality at 28°C, but not at high temperature (34°C) indicating lethality was suppressed. When hybrid seedlings cultured at 34°C were transferred to 28°C, lethal symptoms appeared. Chromatin condensation and nuclear fragmentation were observed in leaf protoplasts isolated from hybrid seedlings expressing lethality. Agarose gel analysis of DNA extracted from leaves of hybrid seedlings revealed a specific ladder pattern, suggesting nucleosomal fragmentation. Nuclear fragmentation was correlated with lethal symptoms in hybrid seedlings, as confirmed by flow cytometry. These results were observed in hybrid seedlings transferred to 28°C from 34°C but not in hybrid seedlings maintained at 34°C. This phenomenon suggests that apoptosis in hybrid seedlings from the cross N. debneyi×N. tabacum is temperature dependent.

Molecular and Cytogenetic Characterization of Triploid Somatic Hybrids between 'Shogun' Mandarin and Grapefruit.

Somatic hybrids were produced between embryogenic callus protoplasts of ‘Shogun’ mandarin (Citrus reticulata Blanco, 2n = 2x = 18) and leaf protoplasts of grapefruit (C. paradisi Macf., 2n = 2x =18) by electrofusion. Hybridity of the two regenerated plants was confirmed by leaf characteristics and nuclear genome analysis using RAPD and PCR-RFLP. Flow cytometry and root-tip cell count of the two hybrids revealed a chromosome number of 27. These triploid somatic hybrids on transfer to field showed normal morphology and vigorous growth. Field performances as well as disease resistance are under investigation.

The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells.

The transpiration stream that passes through a plant may follow an apoplastic route, with low resistance to flow, or a cell-to-cell route, in which cellular membranes impede water flow. However, passage of water through membranes can be facilitated by aquaporins thereby decreasing resistance. We investigated the relationship between transpiration, which can be down-regulated by abscisic acid (ABA) or by high humidity, and the osmotic water permeability (P(os)) of protoplasts. By using leaf protoplasts of wild-type (wt) Arabidopsis thaliana plants and of mutants that are low in ABA (aba1) or insensitive to ABA (abi1 and abi2), we found that protoplasts from aba1 and abi mutants have very low P(os) values compared with those from wt plants when the plants are grown at 45% relative humidity. High values of P(os) were found 3 h after the addition of ABA to the culture medium of aba1 plants; addition of ABA to abi plants did not restore the P(os) to wt levels. There was no such increase in P(os) when excised leaves of aba1 plants were treated with ABA. When the transpiration stream was attenuated by growing the plants at 85% relative humidity, the P(os) of protoplasts from all plants (wt and mutants) was higher. We suggest that attenuation of the transpiration stream in whole plants is required for the up-regulation of the P(os) of the membranes, and that this up-regulation, which does not require ABA, is mediated by the activation of aquaporins in the plasma membrane.

Control of the glutathione S‐transferase and mas1′ promoter‐driven GUS activity in auxin heterotrophic and autotrophic tobacco calli by exogenous 2,4‐d‐induced ethylene

Auxin autotrophic and heterotrophic lines of tobacco calli may differ not only in their indoleacetic acid (IAA) synthetizing abilities and sensitivities to exogenous auxins, but also in their gene expression patterns. Auxin autotrophic callus tissues from the leaf protoplasts of transgenic Nicotiana tabacum SR1 plants involving mas1′::GUS gene fusion were generated and the growth of cultures was compared with that of the heterotrophic lines of the same transgenic tissues on MS medium containing different concentrations of IAA or 2,4‐d. The mas1′::GUS gene fusion expression was investigated, together with the glutathione S‐transferase activities (GST, EC 2.5.1.18) in auxin autotrophic and heterotrophic tobacco calli. Both the mas1′ promoter and GST gene promoters contain ocs or ocs‐like elements, responsible for both auxin and ethylene/wound inducibility. The mas1′ promoter exhibited a much higher expression activity in the heterotrophic cultures growing on IAA than in the autotrophic ones, but in contrast with the natural auxin, the mas1′::GUS activity decreased at elevated 2,4‐d concentrations in the heterotrophic tissues and increased with increasing 2,4‐d concentrations in the autotrophic lines. The induction of GST activity by different exogenous auxin concentrations was much higher in the autotrophic lines, especially in the case of 2,4‐d. Higher concentrations of external 2,4‐d resulted in increased ethylene production, which displayed different kinetics in the two types of calli. The ethylene‐inducing 2,4‐d concentrations increased the growth of the heterotrophic, but decreased that of the autotrophic lines. Blocking the ethylene receptors and hence the signal perception by 2,5‐norbornadiene (NBD) in the heterotrophic tissues increased the 2,4‐d‐induced mas1′ promoter and GST activities, suggesting that the gaseous hormone counteracted the auxin response pathway. This was not found in the autotrophic tissues, where NBD decreased the mas1′‐driven GUS activity. The GST activities were slightly decreased, or almost independent of the action of ethylene. It is suggested that the cross‐talk between the auxin‐ and ethylene‐induced signal transduction pathways may differ in the auxin autotrophic and heterotrophic lines.

Novel anther-specific myb genes from tobacco as putative regulators of phenylalanine ammonia-lyase expression.

Two cDNA clones (NtmybAS1 and NtmybAS2) encoding MYB-related proteins with strong sequence similarity to petunia (Petunia hybrida) PhMYB3 were isolated from a tobacco (Nicotiana tabacum cv Samsun) pollen cDNA library. Northern blot and in situ hybridization revealed that NtmybAS transcripts are specifically expressed in both sporophytic and gametophytic tissues of the anther including tapetum, stomium, vascular tissue, and developing pollen. Random binding site selection assays revealed that NtMYBAS1 bound to DNA sequences closely resembling consensus MYB binding sites MBSI and MBSIIG, with a higher affinity for MBSI. Transient expression analyses of the N-terminal MYB domain demonstrated the presence of functional nuclear localization signals, and full-length NtMYBAS1 was able to activate two different phenylalanine ammonia-lyase promoters (PALA and gPAL1) in tobacco leaf protoplasts. Similar analysis of truncated NtmybAS1 cDNAs identified an essential, C-terminal trans-activation domain. Further in situ hybridization analyses demonstrated strict co-expression of NtmybAS and gPAL1 in the tapetum and stomium. Despite abundant expression of NtmybAS transcripts in mature pollen, gPAL1 transcripts were not detectable in pollen. Our data demonstrate that NtMYBAS1 is a functional anther-specific transcription factor, which is likely to be a positive regulator of gPAL1 expression and phenylpropanoid synthesis in sporophytic, but not in gametophytic, tissues of the anther.