A novel role of BPCs in the control of medial domain differentiation during gynoecium development in Arabidopsis thaliana

Basic pentacysteine (BPC) transcription factors have been identified in a large variety of plant species. In Arabidopsis thaliana there are seven BPC genes, which, except for BPC5, are expressed ubiquitously. BPC genes are functionally redundant in a wide range of developmental processes. Recently, we reported that BPC1 binds to guanine and adenine (GA)-rich consensus sequences in the seedstick (STK) promoter in vitro and induces conformational changes. Here we show by chromatin immunoprecipitation experiments that in vivo BPCs also bind to the consensus boxes, and when these were mutated, expression from the STK promoter was derepressed, resulting in ectopic expression in the inflorescence. We also reveal that short vegetative phase (SVP) is a direct regulator of STK. SVP is a floral meristem identity gene belonging to the MADS box gene family. The SVP-APETALA1 (AP1) dimer recruits the SEUSS (SEU)-LEUNIG (LUG) transcriptional cosuppressor to repress floral homeotic gene expression in the floral meristem. Interestingly, we found that GA consensus sequences in the STK promoter to which BPCs bind are essential for recruitment of the corepressor complex to this promoter. Our data suggest that we have identified a new regulatory mechanism controlling plant gene expression that is probably generally used, when considering BPCs' wide expression profile and the frequent presence of consensus binding sites in plant promoters.

Diverse roles for a class II BPC gene in Camellia japonica through tissue-specific regulation of gene expression.

Plant transcription factors generally act in complex regulatory networks that function at multiple levels to govern plant developmental programs. Dissection of the interconnections among different classes of transcription factors can elucidate these regulatory networks and thus improve our understanding of plant development. Here, we investigated the molecular and functional relationships of the transcription factors ABSCISIC ACID INSENSITIVE 4 (ABI4) and members of the BASIC PENTACYSTEINE (BPC) family in lateral root (LR) development of Arabidopsis thaliana. Genetic analysis showed that BPCs promote LR development by repressing ABI4 expression. Molecular analysis showed that BPCs bind to the ABI4 promoter and repress ABI4 transcription in roots. BPCs directly recruit the Polycomb Repressive Complex 2 (PRC2) to the ABI4 locus and epigenetically repress ABI4 expression by catalyzing the trimethylation of histone H3 at Lys27. In addition, BPCs and ABI4 co-ordinate their activities to fine-tune the levels of PIN-FORMED1, a component of the auxin signaling pathway, and thus modulate LR formation. These results establish a functional relationship between two universal and multiple-role transcription factors, and provide insight into the mechanisms of the transcriptional regulatory networks that affect Arabidopsis organogenesis.

GAGA-binding proteins in plants are encoded by the BARLEY B-RECOMBINANT / BASIC PENTACYSTEINE (BBR/BPC) family, which can be spilt into several groups on the basis of sequence divergence. The proteins of the different groups share an evolutionary conserved BASIC PENTACYSTEINE (BPC) domain at their very C-terminus that is important for DNA binding. Hallmark of this domain are five Cysteines at defined positions and spacing, which are considered to form a zinc-finger like structure that is involved in GAGA-motif recognition. Here, we report the formation of stabile homodimers between Arabidopsis thaliana group I member BPC1 or between group II member BPC6 in SDS-PAGE. Serial mutations of the highly conserved five Cysteines in the BPC domain of Arabidopsis thaliana BPC1 were tested for their capacity to bind to GAGA-motifs by DPI-ELISA. Our results do not support the idea of a direct involvement of these residues in making physical contact with the DNA, e.g. by formation of a zinc-finger structure. Instead, the data implies an indispensable function for the five Cysteines in homodimerization and stabilization of the protein structure by disulfide bonds. Accordingly, protein folding and structure prediction suggests the formation of a scaffold for dimerization that is supported by three intermolecular and one intramolecular S-S bond. The high degree of conservation between the BPC domains from the different groups and from different species denotes that this role for the five Cysteines might be evolutionary retained.

In Arabidopsis, the gynoecium, the inner whorl of the flower, is the female reproductive part. Many tissues important for fertilization such as the stigma, style, transmitting tract, placenta, ovules, and septum, comprising the medial domain, arise from the carpel margin meristem. During gynoecium development, septum fusion occurs and tissues form continuously to prepare for a successful pollination and fertilization. During gynoecium development, cell wall modifications take place and one of the most important is the formation of the transmitting tract, having a great impact on reproductive competence because it facilitates pollen tube growth and movement through the ovary. In this study, using a combination of classical staining methods, fluorescent dyes, and indirect immunolocalization, we analyzed cell wall composition and modifications accompanying medial domain formation during gynoecium development. We detected coordinated changes in polysaccharide distribution through time, cell wall modifications preceding the formation of the transmitting tract, mucosubstances increase during transmitting tract formation, and a decrease of mannan distribution. Furthermore, we also detected changes in lipid distribution during septum fusion. Proper cell wall composition and modifications are important for postgenital fusion of the carpel (septum fusion) and transmitting tract formation, because these tissues affect plant reproductive competence.

Spatiotemporal regulation of gene expression is critical for proper developmental timing in plants and animals. The transcription factor FUSCA3 (FUS3) regulates developmental phase transitions by acting as a link between hormonal pathways in Arabidopsis (Arabidopsis thaliana). However, the mechanisms governing its spatiotemporal expression pattern are poorly understood. Here, we show that FUS3 is repressed in the ovule integuments and seed endosperm. FUS3 repression requires class I BASIC PENTACYSTEINE (BPC) proteins, which directly bind GA/CT cis-elements in FUS3 and restrict its expression pattern. During vegetative and reproductive development, FUS3 derepression in bpc1-1 bpc2 (bpc1/2) double mutant or misexpression in ProML1:FUS3 lines causes dwarf plants carrying defective flowers and aborted ovules. After fertilization, ectopic FUS3 expression in bpc1/2 endosperm or ProML1:FUS3 endosperm and endothelium increases endosperm nuclei proliferation and seed size, causing delayed or arrested embryo development. These phenotypes are rescued in bpc1/2 fus3-3 Finally, class I BPCs interact with FIS-PRC2 (FERTILIZATION-INDEPENDENT SEED-Polycomb Repressive Complex2), which represses FUS3 in the endosperm during early seed development. We propose that BPC1 and 2 promote the transition from reproductive to seed development by repressing FUS3 in ovule integuments. After fertilization, BPC1 and 2 and FIS-PRC2 repress FUS3 in the endosperm to coordinate early endosperm and embryo growth.

BASIC PENTACYSTEINE (BPC) is a small family of plant-specific transcription factors that play crucial roles in plant growth, development processes, and response to abiotic stresses. However, the specific roles of Nicotiana tabacum BPCs (NtBPCs) remain ambiguous. Here, we identified 12 NtBPC genes, 5 of which were mapped to four chromosomes. Phylogenetic analysis classified these genes into three subfamilies. Collinearity was observed among BPC genes of N. tabacum, Capsicum annuum, and Solanum lycopersicum. Moreover, polypeptides encoded by NtBPC genes within the same subfamily shared similar conserved motifs and protein domains. Subcellular localization showed that 10 NtBPC proteins are localized in the nucleus. Promoter analysis revealed the presence of abiotic stress response elements in the promoters of NtBPCs. Further tissue-specific expression analysis using RT-qPCR revealed that NtBPCs are highly expressed in stems and leaves. After drought, NaCl, and cold treatments, NtBPCs exhibited varied expression patterns. These findings provide valuable insights into the evolutionary dynamics of the NtBPC gene family and lay the groundwork for subsequent investigations into the functions of NtBPC genes.

BASIC PENTACYSTEINE (BPC) transcription factors are essential regulators of plant growth and development. However, BPC functions and the related molecular mechanisms during cucumber (Cucumis sativus L.) responses to abiotic stresses, especially salt stress, remain unknown. We previously determined that salt stress induces CsBPC expression in cucumber. In this study, Csbpc2 transgene-free cucumber plants were created using a CRISPR/Cas9-mediated editing system to explore CsBPC functions associated with the salt stress response. The Csbpc2 mutants had a hypersensitive phenotype, with increased leaf chlorosis, decreased biomass, and increased malondialdehyde and electrolytic leakage levels under salt stress conditions. Additionally, a mutated CsBPC2 resulted in decreased proline and soluble sugar contents and antioxidant enzyme activities, which led to the accumulation of hydrogen peroxide and superoxide radicals. Furthermore, the mutation to CsBPC2 inhibited salinity-induced PM-H+-ATPase and V-H+-ATPase activities, resulting in decreased Na+ efflux and increased K+ efflux. These findings suggest that CsBPC2 may mediate plant salt stress resistance through its effects on osmoregulation, reactive oxygen species scavenging, and ion homeostasis-related regulatory pathways. However, CsBPC2 also affected ABA signaling. The mutation to CsBPC2 adversely affected salt-induced ABA biosynthesis and the expression of ABA signaling-related genes. Our results indicate that CsBPC2 may enhance the cucumber response to salt stress. It may also function as an important regulator of ABA biosynthesis and signal transduction. These findings will enrich our understanding of the biological functions of BPCs, especially their roles in abiotic stress responses, thereby providing the theoretical basis for improving crop salt tolerance.

-We studied within-season mate switching in two populations of House Wrens (Troglodytes aedon) in central Illinois over nine breeding seasons. On the East Bay site, 35.2% of the monogamous pairs switched mates. On this less-preferred habitat, there were fewer potential mates. In contrast, on the Mackinaw site, 58.8% of the monogamous pairs switched mates, and there were potential mates available throughout the breeding season. There was no consistent immediate reproductive cost or benefit to mate switching. We suggest that mate switching in these House Wren populations is a consequence of varying mate availability and differing gender-related costs of facultative polygyny and territoriality. Because females incubate the eggs and brood the chicks, males have the first opportunity to desert their mate and offspring, which often results in polygynous matings. After the chicks leave the nest, males that feed fledglings may lose their territory and the opportunity to breed again. Females do not incur such a cost, and they easily move to another territory and another mate. Frequencies and relative costs and benefits of mate switching also varied considerably among three other House Wren populations. This reveals considerable flexibility in response to different demographic and environmental situations. Knowledge of the basis for this variability is important to understand mate choice in birds. Received 14 February 1990, accepted 14 July 1990. THE DEGREE to which mated birds maintain the pair bond between breeding attempts within a single breeding season varies among species and among populations within a species. Multibrooded passerines often display almost complete mate fidelity, with the male caring for the offspring while his mate begins the next nest (see reviews in Nice 1930, Burns 1983). In these species, the advantages of mate fidelity (e.g. familiarity of mate and territory) outweigh the advantages of mate switching (finding a better mate or territory). Some species that regularly switch mates within seasons include European Starlings (Sturnus vulgaris; Feare and Burnham 1978), Prairie Warblers (Dendroica discolor; Nolan 1978), Indigo Buntings (Passerina cyanea; Carey and Nolan 1979), Winter Wrens (Troglodytes troglodytes; Garson 1980), Barn Swallows (Hirundo rustica; Shields 1984), Song Sparrows (Melodia melospiza; Weatherhead and Boak 1986), Fan-tailed Cisticola (Cisticola juncidis; Ueda 1986), and Pinon Jays (Gymnorhinus cyanocephalus; Marzluff and Balda 1988). The decision of a pair to separate will depend upon the relative costs and benefits of switching to a new partner, and upon mate availability (Maynard Smith 1977). If no potential mates are available, a bird must choose between breeding with its previous mate or not breeding. If new potential mates are available, birds that suffer from low reproductive success at their first nest may have higher subsequent reproductive success if they switch mates. Thus, if there is a direct relationship between immediate reproductive success and mate switching, birds with failed nests should be more likely to switch mates than those with successful nests, and birds that switch mates after nest failure should be more successful subsequently than those not switching. Birds switch mates for various reasons. They may be attempting to increase their reproductive success by finding a more experienced, older mate. They may be attempting to minimize the time between nesting attempts, or to obtain a higher-quality territory (Shields 1984). Switching to an older, more experienced mate may result in higher reproductive success because, in some species, more experienced or older birds have higher reproductive success than less experienced, younger individuals (Rowley 1983). If so, the frequency of mate switching should be lowest in old/experienced pairs, intermediate in mixed old/experienced-young/ inexperienced pairs, and highest in young/inexperienced pairs. Switching could also increase reproductive success by minimizing the time between nesting attempts, if breeding seasons are short or reproductive success decreases as the breeding season progresses (Burns 1983). 60 The Auk 108: 60-70. January 1991 This content downloaded from 157.55.39.51 on Wed, 10 Aug 2016 05:35:54 UTC All use subject to http://about.jstor.org/terms January 1991] Mate Switching in House Wrens 61 In this case, if one parent can raise at least some of the brood alone, the other parent should abandon the first brood and switch to another

Gynoecium development is dependent on gene regulation and hormonal pathway interactions. The phytohormones auxin and cytokinin are involved in many developmental programs, where cytokinin is normally important for cell division and meristem activity, while auxin induces cell differentiation and organ initiation in the shoot. The MADS-box transcription factor AGAMOUS (AG) is important for the development of the reproductive structures of the flower. Here, we focus on the relationship between AG and cytokinin in Arabidopsis thaliana, and use the weak ag-12 and the strong ag-1 allele. We found that cytokinin induces carpeloid features in an AG-dependent manner and the expression of the transcription factors CRC, SHP2, and SPT that are involved in carpel development. AG is important for gynoecium development, and contributes to regulating, or else directly regulates CRC, SHP2, and SPT. All four genes respond to either reduced or induced cytokinin signaling and have the potential to be regulated by cytokinin via the type-B ARR proteins. We generated a model of a gene regulatory network, where cytokinin signaling is mainly upstream and in parallel with AG activity.

The gynoecium is the female reproductive structure in flowering plants. It has been known that the CLAVATA1 (CLV1) gene regulates the floral meristem maintenance and floral organ number in Arabidopsis thaliana. However, whether CLV1 plays roles in gynoecium development remains uncharacterized. In this study, we showed that CLV1 genetically interacts with Unusual Floral Organs (UFO) to regulate the development of Arabidopsis gynoecium. Through forward genetic screen, we identified a clv1 allele, clv1-9, showing abnormal gynoecium development. Some epidermal cells on the growing inner surfaces of the clv1-9 gynoecium failed to fuse after contacting with each other, suggesting that CLV1 is essential for proper postgenital fusion. Furthermore, the ufo clv1-9 double mutant exhibited an expanded upper gynoecium, narrowed lower part of the ovary, altered vascular patterning and abaxial outward proliferation of the transmitting tract tissues. The transcription level of the ETT gene, which is required for gynoecium structure and pattern formation, was down-regulated in the ufo clv1-9 double mutant genoecia. Taken together, our results indicate that CLV1 functions together with UFO to regulate gynoecium development likely through modulating the expression of ETT.

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y-specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y-gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.

Hormonal control of the development of the gynoecium

In plants, correct formation of reproductive organs is critical for successful seedset and perpetuation of the species. Plants have evolved different molecular mechanisms to coordinate flower and seed development at the proper time of the year. Among the plant-specific RELATED TO ABI3 AND VP1 (RAV) family of transcription factors, only TEMPRANILLO1 (TEM1) and TEM2 have been shown to affect reproductive development in Arabidopsis (Arabidopsis thaliana). They negatively regulate the floral transition through direct repression of FLOWERING LOCUS T and GIBBERELLIN 3-OXIDASE1/2, encoding major components of the florigen. Here we identify RAV genes from rice (Oryza sativa), and unravel their regulatory roles in key steps of reproductive development. Our data strongly suggest that, like TEMs, OsRAV9/OsTEM1 has a conserved function as a repressor of photoperiodic flowering upstream of the floral activators OsMADS14 and Hd3a, through a mechanism reminiscent of that one underlying floral transition in temperate cereals. Furthermore, OsRAV11 and OsRAV12 may have acquired a new function in the differentiation of the carpel and the control of seed size, acting downstream of floral homeotic factors. Alternatively, this function may have been lost in Arabidopsis. Our data reveal conservation of RAV gene function in the regulation of flowering time in monocotyledonous and dicotyledonous plants, but also unveil roles in the development of rice gynoecium.

Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA Affects Developmental Timing and Patterning in Arabidopsis