HDF1, a novel flowering time regulator identified in a mutant suppressing sensitivity to red light reduced 1 early flowering.

Arabidopsis SENSITIVITY TO RED LIGHT REDUCED 1 (SRR1) delays the transition from vegetative to reproductive development in noninductive conditions. A second-site suppressor screen for novel genes that overcome early flowering of srr1-1 identified a range of suppressor of srr1-1 mutants flowering later than srr1-1 in short photoperiods. Here, we focus on mutants flowering with leaf numbers intermediate between srr1-1 and Col. Ssm67 overcomes srr1-1 early flowering independently of day-length and ambient temperature. Full-genome sequencing and linkage mapping identified a causative SNP in a gene encoding a Haloacid dehalogenase superfamily protein, named HAD-FAMILY REGULATOR OF DEVELOPMENT AND FLOWERING 1 (HDF1). Both, ssm67 and hdf1-1 show increased levels of FLC, indicating that HDF1 is a novel regulator of this floral repressor. HDF1 regulates flowering largely independent of SRR1, as the effect is visible in srr1-1 and in Col, but full activity on FLC may require SRR1. Furthermore, srr1-1 has a delayed leaf initiation rate that is dependent on HDF1, suggesting that SRR1 and HDF1 act together in leaf initiation. Another mutant flowering intermediate between srr1-1 and wt, ssm15, was identified as a new allele of ARABIDOPSIS SUMO PROTEASE 1, previously implicated in the regulation of FLC stability.

SYL3-k increases style length and yield of F1 seeds via enhancement of endogenous GA4 content in Oryza sativa L. pistils.

KEY MESSAGE: SYL3-k allele increases the outcrossing rate of male sterile line and the yield of hybrid F1 seeds via enhancement of endogenous GA4 content in Oryza sativa L. pistils. The change in style length might be an adaptation of rice cultivation from south to north in the northern hemisphere. The style length (SYL) in rice is one of the major factors influencing the stigma exertion, which affects the outcross rate of male sterile line and the yield of hybrid F1 seeds. However, the biological mechanisms underlying SYL elongation remain elusive. Here, we report a map-based cloning and characterisation of the allele qSYL3-k. The qSYL3-k allele encodes a MADS-box family transcription factor, and it is expressed in various rice organs. The qSYL3-k allele increases SYL via the elongation of cell length in the style, which is associated with a higher GA4 content in the pistil. The expression level of OsGA3ox2 in pistils with qSYL3-k alleles is significantly higher than that in pistils with qSYL3-n allele on the same genome background of Nipponbare. The yield of F1 seeds harvested from plants with 7001SSYL3-k alleles was 16% higher than that from plants with 7001SSYL3-n allele. The sequence data at the qSYL3 locus in 136 accessions showed that alleles containing the haplotypes qSYL3AA, qSYL3AG, and qSYL3GA increased SYL, whereas those containing the haplotype qSYL3GG decreased it. The frequency of the haplotype qSYL3GG increases gradually from the south to north in the northern hemisphere. These findings will facilitate improvement in SYL and yield of F1 seeds henceforward.

Genetic Interaction of SEEDSTICK, GORDITA and AUXIN RESPONSE FACTOR 2 during Seed Development.

Seed development is under the control of complex and coordinated molecular networks required for the formation of its different components. The seed coat development largely determines final seed size and shape, in addition to playing a crucial role in protecting the embryo and promoting germination. In this study, we investigated the role of three transcription factors known to be active during seed development in Arabidopsis thaliana: SEEDSTICK (STK) and GORDITA (GOA), two MADS-domain proteins, and AUXIN RESPONSE FACTOR 2 (ARF2), belonging to the ARF family. Through a reverse genetic approach, we characterized the seed phenotypes of all the single, double and triple loss-of-function mutants in relation to seed size/shape and the effects on metabolic pathways occurring in the seed coat. This approach revealed that dynamic networks involving these TFs are active throughout ovule and seed development, affecting the formation of the seed coat. Notably, while the genetic interaction among these genes results in synergies that control the promotion of cell expansion in the seed coat upon pollination and production of proanthocyanidins, functional antagonists arise in the control of cell proliferation and release of mucilage.

Nitrogen Signaling Genes and SOC1 Determine the Flowering Time in a Reciprocal Negative Feedback Loop in Chinese Cabbage (Brassica rapa L.) Based on CRISPR/Cas9-Mediated Mutagenesis of Multiple BrSOC1 Homologs.

Precise flowering timing is critical for the plant life cycle. Here, we examined the molecular mechanisms and regulatory network associated with flowering in Chinese cabbage (Brassica rapa L.) by comparative transcriptome profiling of two Chinese cabbage inbred lines, "4004" (early bolting) and "50" (late bolting). RNA-Seq and quantitative reverse transcription PCR (qPCR) analyses showed that two positive nitric oxide (NO) signaling regulator genes, nitrite reductase (BrNIR) and nitrate reductase (BrNIA), were up-regulated in line "50" with or without vernalization. In agreement with the transcription analysis, the shoots in line "50" had substantially higher nitrogen levels than those in "4004". Upon vernalization, the flowering repressor gene Circadian 1 (BrCIR1) was significantly up-regulated in line "50", whereas the flowering enhancer genes named SUPPRESSOR OF OVEREXPRESSION OF CONSTANCE 1 homologs (BrSOC1s) were substantially up-regulated in line "4004". CRISPR/Cas9-mediated mutagenesis in Chinese cabbage demonstrated that the BrSOC1-1/1-2/1-3 genes were involved in late flowering, and their expression was mutually exclusive with that of the nitrogen signaling genes. Thus, we identified two flowering mechanisms in Chinese cabbage: a reciprocal negative feedback loop between nitrogen signaling genes (BrNIA1 and BrNIR1) and BrSOC1s to control flowering time and positive feedback control of the expression of BrSOC1s.

Molecular cloning and potential role of DiSOC1s in flowering regulation in Davidia involucrata Baill.

Davidia involucrata Baill. (dove tree) is unique Tertiary relic plant in China, also known as 'living fossil' and 'giant panda'. The MADS-box family gene SOC1 is involved in the regulatory pathway that integrates flowering signals to promote flowering at the optimal time. In this study, we isolated and identified two dove tree SOC1 homologues, named DiSOC1-a and DiSOC1-b. These two sequences possess highly conserved domains MADS-box and SOC1-motif, as well as the semi-conserved region K-box. DiSOC1-a and DiSOC1-b were expressed at varying levels in all tested tissues of dove tree and shared high levels of expression in the flower buds. The expression tendencies of both genes in bract were initially upward and then downward and were highest in young bracts. Neither DiSOC1-a nor DiSOC1-b was expressed in immature leaves. Proteins encoded by DiSOC1-a and DiSOC1-b were located in the nucleus. In addition, ectopic overexpression of both genes in WT Arabidopsis promoted early flowering and the growth of the main bolt. Taken together, these results suggest that DiSOC1-a and DiSOC1-b are involved in the flowering initiation and the main bolt growth process of dove tree. Our results provide a foundation for horticultural breeding to control flowering time of dove tree.

The Transcriptome of Cunninghamia lanceolata male/female cone reveal the association between MIKC MADS-box genes and reproductive organs development.

BACKGROUND: Cunninghamia lanceolata (Chinese fir), a member of the conifer family Cupressaceae, is one of the most popular cultivated trees for wood production in China. Continuous research is being performed to improve C. lanceolata breeding values. Given the high rate of seed abortion (one of the reasons being the failure of ovule and pollen development) in C. lanceolata, the proper formation of female/male cones could theoretically increase the number of offspring in future generations. MIKC MADS-box genes are well-known for their roles in the flower/cone development and comprise the typical/atypical floral development model for both angiosperms and gymnosperms. RESULTS: We performed a transcriptomic analysis to find genes differentially expressed between female and male cones at a single, carefully determined developmental stage, focusing on the MIKC MADS-box genes. We finally obtained 47 unique MIKC MADS-box genes from C. lanceolata and divided these genes into separate branches. 27 out of the 47 MIKC MADS-box genes showed differential expression between female and male cones, and most of them were not expressed in leaves. Out of these 27 genes, most B-class genes (AP3/PI) were up-regulated in the male cone, while TM8 genes were up-regulated in the female cone. Then, with no obvious overall preference for AG (class C + D) genes in female/male cones, it seems likely that these genes are involved in the development of both cones. Finally, a small number of genes such as GGM7, SVP, AGL15, that were specifically expressed in female/male cones, making them candidate genes for sex-specific cone development. CONCLUSIONS: Our study identified a number of MIKC MADS-box genes showing differential expression between female and male cones in C. lanceolata, illustrating a potential link of these genes with C. lanceolata cone development. On the basis of this, we postulated a possible cone development model for C. lanceolata. The gene expression library showing differential expression between female and male cones shown here, can be used to discover unknown regulatory networks related to sex-specific cone development in the future.

Genome-wide identification of MADS-box gene family in sacred lotus (Nelumbo nucifera) identifies a SEPALLATA homolog gene involved in floral development.

BACKGROUND: Sacred lotus (Nelumbo nucifera) is a vital perennial aquatic ornamental plant. Its flower shape determines the horticultural and ornamental values. However, the mechanisms underlying lotus flower development are still elusive. MADS-box transcription factors are crucial in various features of plant development, especially in floral organogenesis and specification. It is still unknown how the MADS-box transcription factors regulate the floral organogenesis in lotus. RESULTS: To obtain a comprehensive insight into the functions of MADS-box genes in sacred lotus flower development, we systematically characterized members of this gene family based on the available genome information. A total of 44 MADS-box genes were identified, of which 16 type I and 28 type II genes were categorized based on the phylogenetic analysis. Furthermore, the structure of MADS-box genes and their expressional patterns were also systematically analyzed. Additionally, subcellular localization analysis showed that they are mainly localized in the nucleus, of which a SEPALLATA3 (SEP3) homolog NnMADS14 was proven to be involved in the floral organogenesis. CONCLUSION: These results provide some fundamental information about the MADS-box gene family and their functions, which might be helpful in not only understanding the mechanisms of floral organogenesis but also breeding of high ornamental value cultivars in lotus.

Maternal transmission of the epigenetic 'memory of winter cold' in Arabidopsis.

Some plants can 'remember' past environmental experience to become adapted to a given environment. For instance, after experiencing prolonged low-temperature exposure in winter (winter cold), vernalization-responsive plants remember past cold experience when temperature rises in spring, to acquire competence to flower at a later season favourable for seed production1,2. In Arabidopsis thaliana, prolonged cold induces silencing of the potent floral repressor FLOWERING LOCUS C (FLC) by Polycomb group (PcG) chromatin modifiers. This Polycomb-repressed chromatin state is epigenetically maintained and thus 'memorized' in subsequent growth and development upon return to warmth1,3. 'Memory of winter cold' has been viewed as being mitotically stable but meiotically unstable3-5, and thus not to be transmitted intergenerationally. In general, whether and how chromatin-mediated environmental memories are transmitted across generations are unknown in plants. Here, we show that the cold-induced Polycomb-repressed chromatin state at FLC or memory of winter cold is maintained in the egg cell, that is meiotically stable in the process of female gamete formation, and provide evidence that this Polycomb-mediated memory is not maintained in the sperm cell. Moreover, we show that this cold memory is inherited maternally but not paternally to the zygote and early embryos. Our study demonstrates and further provides mechanistic insights into intergenerational transmission of chromatin state-mediated environmental memories in plants.

Identification, characterization and functional analysis of AGAMOUS subfamily genes associated with floral organs and seed development in Marigold (Tagetes erecta).

BACKGROUND: AGAMOUS (AG) subfamily genes regulate the floral organs initiation and development, fruit and seed development. At present, there has been insufficient study of the function of AG subfamily genes in Asteraceae. Marigold (Tagetes erecta) belongs to Asteraceae family whose unique inflorescence structure makes it an important research target for understanding floral organ development in plants. RESULTS: Four AG subfamily genes of marigold were isolated and phylogenetically grouped into class C (TeAG1 and TeAG2) and class D (TeAGL11-1 and TeAGL11-2) genes. Expression profile analysis demonstrated that these four genes were highly expressed in reproductive organs of marigold. Subcellular localization analysis suggested that all these four proteins were located in the nucleus. Protein-protein interactions analysis indicated that class C proteins had a wider interaction manner than class D proteins. Function analysis of ectopic expression in Arabidopsis thaliana revealed that TeAG1 displayed a C function specifying the stamen identity and carpel identity, and that TeAGL11-1 exhibited a D function regulating seed development and petal development. In addition, overexpression of both TeAG1 and TeAGL11-1 leaded to curling rosette leaf and early flowering in Arabidopsis thaliana. CONCLUSIONS: This study provides an insight into molecular mechanism of AG subfamily genes in Asteraceae species and technical support for improvement of several floral traits.

Cloning of a SEPALLATA4-like gene (IiSEP4) in Isatis indigotica Fortune and characterization of its function in Arabidopsis thaliana.

E-class MADS-box genes, SEPALLATA (SEP), participate in various aspects of plant development together with B-, C- and D-class MADS-box genes. IiSEP4, a homologous gene of SEP4, was cloned from Isatis indigotica. IiSEP4 was highly expressed in sepals, and its mRNA was mildly detected in leaves, inflorescences, flowers, stamens and young silicles. Constitutive expression of IiSEP4 in Arabidopsis thaliana caused early flowering, accompanied by the reduction of flowers and floral organs. Moreover, the sepals in some flowers were transformed into carpelloid structures with stigmatic papillae, and obviously accompanied by ovule formation. Yeast two-hybrid assays demonstrated that IiSEP4 interacts with other woad MADS proteins to determine the identity of floral organs. These findings reveal the important roles of IiSEP4 in floral development of I. indigotica. The results of this study can lay a foundation for further study on biological functions of MADS transcriptional factors in I. indigotica.

Homeotic transformation from stamen to petal in Eriobotrya japonica is associated with hormone signal transduction and reduction of the transcriptional activity of EjAG.

Double-flower loquat (Eriobotrya japonica) is a new germplasm with homeotic transformation of stamen into petal in whorl 3. However, little information is available on the molecular mechanism of this transformation. Herein, we analyzed the transcriptome, candidate genes and endogenous hormones to investigate the mechanisms underlying this homeotic transformation. Some transcription factors, such as MADS-box, TCP and MYB, were significantly differentially expressed. Importantly, we confirmed that one of these (DN39625_c0_g1), which encoded a C-class floral homeotic protein referred to as AGAMOUS ortholog (EjAG), was significantly downregulated. Subcellular localization of EjAG was found to be in the nucleus. Ectopic expression of EjAG rescued the development of stamens and carpels from the double-flower phenotype in an Arabidopsis ag mutant, suggesting that EjAG expression is associated with double-flower formation. Meanwhile, enrichment analyses showed that the differentially expressed genes (DEGs) were mainly involved in the metabolic pathways of hormone signal transduction. The DEGs of auxin, gibberellin A (GA) and cytokinin signaling pathways were mainly upregulated. However, the DEGs of abscisic acid (ABA) and the ethylene signaling pathway were mainly downregulated. Accordingly, the concentrations of indoleacetic acid, kinetin and GA3 were high at the petaloid stamen stage, but the ABA concentration remained low. The identified genes and pathways provide abundant sequence resources for studying the mechanisms underlying the homeotic transformation in loquat and other Rosaceae species.

Fine mapping of Brassica napus blackleg resistance gene Rlm1 through bulked segregant RNA sequencing.

The fungal pathogen Leptosphaeria maculans causes blackleg disease on canola and rapeseed (Brassica napus) in many parts of the world. A B. napus cultivar, 'Quinta', has been widely used for the classification of L. maculans into pathogenicity groups. In this study, we confirmed the presence of Rlm1 in a DH line (DH24288) derived from B. napus cultivar 'Quinta'. Rlm1 was located on chromosome A07, between 13.07 to 22.11 Mb, using a BC1 population made from crosses of F1 plants of DH16516 (a susceptible line) x DH24288 with bulked segregant RNA Sequencing (BSR-Seq). Rlm1 was further fine mapped in a 100 kb region from 19.92 to 20.03 Mb in the BC1 population consisting of 1247 plants and a F2 population consisting of 3000 plants using SNP markers identified from BSR-Seq through Kompetitive Allele-Specific PCR (KASP). A potential resistance gene, BnA07G27460D, was identified in this Rlm1 region. BnA07G27460D encodes a serine/threonine dual specificity protein kinase, catalytic domain and is homologous to STN7 in predicted genes of B. rapa and B. oleracea, and A. thaliana. Robust SNP markers associated with Rlm1 were developed, which can assist in introgression of Rlm1 and confirm the presence of Rlm1 gene in canola breeding programs.

Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway.

Flocculation is an essential characteristic of yeast cells required for survival under adverse conditions. The multicellular structure (flocs) of yeast provides a suitable microenvironment to enhance the chances of survival during stress conditions. Although the signaling events triggering flocculation have been studied earlier, molecular mechanisms remain elusive. In the present study, we used flocculating sen1 mutants to identify the mechanism of flocculation. Based on the abnormal cell surface morphology and constitutive phosphorylation of Slt2p in flocculating sen1 mutant cells, we hypothesized if flocculation was regulated by the cell wall integrity (CWI) pathway. Up-regulation of FLO genes in wild-type cells was observed upon the activation of CWI pathway either by chemical treatment or by deleting Slt2 phosphatase (Msg5). Our study with Slt2 mutants reveals that the active state of Slt2 is indispensable for flocculation. Deletion of either SLT2 or RLM1 leads to reduced flocculation. Furthermore, we observed overlapping binding sites for Rlm1 and Tup1 at the promoters of almost all the FLO genes. Finally, we show higher Rlm1 and lower Tup1 occupancy at the promoters of FLO1 and FLO5 in flocculating cells. Altogether we demonstrate that CWI MAPK (Slt2) pathway uses a non-catalytic mechanism to activate the transcription of FLO genes.

Nitrate Transporter 1.1 is involved in regulating flowering time via transcriptional regulation of FLOWERING LOCUS C in Arabidopsis thaliana.

Nitrate Transporter 1.1 (NRT1.1) is a nitrate transporter and sensor that modulates plant metabolism and growth. It has previously been shown that NRT1.1 is involved in the regulation of flowering time in Arabidopsis thaliana. In this study, we mainly used genetic and molecular methods to reveal the key flowering pathway that NRT1.1 may be involved in. Mutant alleles of CO and FLC, two crucial components in the flowering pathway, were introduced into the NRT1.1 defective mutant background by crossing. When the CO mutation was introduced into chl1-5 plants, the double mutant had delayed flowering time, and the CO transcription levels did not change in the chl1-5 plants. These results indicate that the CO-dependent photoperiod may be not associated with the delayed flowering shown by chl1-5. However, FLC loss of function could rescue the late flowering phenotype of the chl1-5 mutant, and FLC expression levels significantly increased in the NRT1.1 defective mutant plants. The FT expression levels in the chl1-5flc-3 double mutant plants recovered when the FLC mutation was introduced into chl1-5 plants and the up-regulation of FLC transcripts in the chl1-5 mutant plants was not related to nitrate availability. Our findings suggest that NRT1.1 affects flowering time via interaction with the FLC-dependent flowering pathway to influence its target gene FT, and that NRT1.1 may be included in an additional signaling pathway that represses the expression of FLC in a nitrate-independent manner.

Transcriptomic analysis of Citrus clementina mandarin fruits maturation reveals a MADS-box transcription factor that might be involved in the regulation of earliness.

BACKGROUND: Harvest time is a relevant economic trait in citrus, and selection of cultivars with different fruit maturity periods has a remarkable impact in the market share. Generation of early- and late-maturing cultivars is an important target for citrus breeders, therefore, generation of knowledge regarding the genetic mechanisms controlling the ripening process and causing the early and late phenotypes is crucial. In this work we analyze the evolution of the transcriptome during fruit ripening in 3 sport mutations derived from the Fina clementine (Citrus clementina) mandarin: Clemenules (CLE), Arrufatina (ARR) and Hernandina (HER) that differ in their harvesting periods. CLE is considered a mid-season cultivar while ARR and HER are early- and late-ripening mutants, respectively. RESULTS: We used RNA-Seq technology to carry out a time course analysis of the transcriptome of the 3 mutations along the ripening period. The results indicated that in these mutants, earliness and lateness during fruit ripening correlated with the advancement or delay in the expression of a set of genes that may be implicated in the maturation process. A detailed analysis of the transcription factors known to be involved in the regulation of fruit ripening identified a member of the MADS box family whose expression was lower in ARR, the early-ripening mutant, and higher in HER, the late-ripening mutant. The pattern of expression of this gene during the maturation period was basically contrary to those of the ethylene biosynthetic genes, SAM and ACC synthases and ACC oxidase. The gene was present in hemizygous dose in the early-ripening mutant. CONCLUSIONS: Our analysis provides new clues about the genetic control of fruit ripening in citrus and allowed the identification of a transcription factor that could be involved in the early phenotype.

Characterization of Maf1 in Arabidopsis: function under stress conditions and regulation by the TOR signaling pathway.

MAIN CONCLUSION: Maf1 repressor activity is critical for plant survival during environmental stresses, and is regulated by its phosphorylation/dephosphorylation through the activity of TOR and PP4/PP2A phosphatases. Maf1 is a global repressor of RNA polymerase III (Pol III), and is conserved in eukaryotes. Pol III synthesizes small RNAs, 5S rRNA, and tRNAs that are essential for protein translation and cell growth. Maf1 is a phosphoprotein and dephosphorylation of Maf1 promotes its repressor activity in yeast and mammals. Plant Maf1 was identified in citrus plants as a canker elicitor-binding protein, and citrus Maf1 represses cell growth associated with canker development. However, functions of plant Maf1 under diverse stress conditions and its regulation by the target of rapamycin (TOR) signaling components are poorly understood. In this study, the Arabidopsis maf1 mutants were more susceptible to diverse stresses and treatment with the TOR inhibitor Torin-1 than wild-type plants. The maf1 mutants expressed higher levels of Maf1 target RNAs, including 5S rRNA and pre-tRNAs in leaf cells, supporting Pol III repressor activity of Arabidopsis Maf1. Cellular stresses and Torin-1 treatment induced dephosphorylation of Maf1, suggesting Maf1 activation under diverse stress conditions. TOR silencing also stimulated Maf1 dephosphorylation, while silencing of catalytic subunit genes of PP4 and PP2A repressed it. Thus, TOR kinase and PP4/PP2A phosphatases appeared to oppositely modulate the Maf1 phosphorylation status. TOR silencing decreased the abundance of the target RNAs, while silencing of the PP4 and PP2A subunit genes increased it, supporting the positive correlation between Maf1 dephosphorylation and its repressor activity. Taken together, these results suggest that repressor activity of Maf1, regulated by the TOR signaling pathway, is critical for plant cell survival during environmental stresses.

An APETALA1 ortholog affects plant architecture and seed yield component in oilseed rape (Brassica napus L.).

BACKGROUND: Increasing the productivity of rapeseed as one of the widely cultivated oil crops in the world is of upmost importance. As flowering time and plant architecture play a key role in the regulation of rapeseed yield, understanding the genetic mechanism underlying these traits can boost the rapeseed breeding. Meristem identity genes are known to have pleiotropic effects on plant architecture and seed yield in various crops. To understand the function of one of the meristem identity genes, APETALA1 (AP1) in rapeseed, we performed phenotypic analysis of TILLING mutants under greenhouse conditions. Three stop codon mutant families carrying a mutation in Bna.AP1.A02 paralog were analyzed for different plant architecture and seed yield-related traits. RESULTS: It was evident that stop codon mutation in the K domain of Bna.AP1.A02 paralog caused significant changes in flower morphology as well as plant architecture related traits like plant height, branch height, and branch number. Furthermore, yield-related traits like seed yield per plant and number of seeds per plants were also significantly altered in the same mutant family. Apart from phenotypic changes, stop codon mutation in K domain of Bna.AP1.A02 paralog also altered the expression of putative downstream target genes like Bna.TFL1 and Bna.FUL in shoot apical meristem (SAM) of rapeseed. Mutant plants carrying stop codon mutations in the COOH domain of Bna.AP1.A02 paralog did not have a significant effect on plant architecture, yield-related traits or the expression of the downstream targets. CONCLUSIONS: We found that Bna.AP1.A02 paralog has pleiotropic effect on plant architecture and yield-related traits in rapeseed. The allele we found in the current study with a beneficial effect on seed yield can be incorporated into rapeseed breeding pool to develop new varieties.

PISTILLATA paralogs in Tarenaya hassleriana have diverged in interaction specificity.

BACKGROUND: Floral organs are specified by MADS-domain transcription factors that act in a combinatorial manner, as summarized in the (A)BCE model. However, this evolutionarily conserved model is in contrast to a remarkable amount of morphological diversity in flowers. One of the mechanisms suggested to contribute to this diversity is duplication of floral MADS-domain transcription factors. Although gene duplication is often followed by loss of one of the copies, sometimes both copies are retained. If both copies are retained they will initially be redundant, providing freedom for one of the paralogs to change function. Here, we examine the evolutionary fate and functional consequences of a transposition event at the base of the Brassicales that resulted in the duplication of the floral regulator PISTILLATA (PI), using Tarenaya hassleriana (Cleomaceae) as a model system. RESULTS: The transposition of a genomic region containing a PI gene led to two paralogs which are located at different positions in the genome. The original PI copy is syntenic in position with most angiosperms, whereas the transposed copy is syntenic with the PI genes in Brassicaceae. The two PI paralogs of T. hassleriana have very similar expression patterns. However, they may have diverged in function, as only one of these PI proteins was able to act heterologously in the first whorl of A. thaliana flowers. We also observed differences in protein complex formation between the two paralogs, and the two paralogs exhibit subtle differences in DNA-binding specificity. Sequence analysis indicates that most of the protein sequence divergence between the two T. hassleriana paralogs emerged in a common ancestor of the Cleomaceae and the Brassicaceae. CONCLUSIONS: We found that the PI paralogs in T. hassleriana have similar expression patterns, but may have diverged at the level of protein function. Data suggest that most protein sequence divergence occurred rapidly, prior to the origin of the Brassicaceae and Cleomaceae. It is tempting to speculate that the interaction specificities of the Brassicaceae-specific PI proteins are different compared to the PI found in other angiosperms. This could lead to PI regulating partly different genes in the Brassicaceae, and ultimately might result in change floral in morphology.

Slt2 MAPK association with chromatin is required for transcriptional activation of Rlm1 dependent genes upon cell wall stress.

The regulation of gene expression through the cell wall integrity (CWI) pathway in yeast is mainly coordinated by the MAPK Slt2 and the transcription factor Rlm1. In this work, we elucidate a new role for Slt2 as a part of the transcriptional activation machinery that regulates CWI gene expression in response to cell wall stress. We show that Slt2 is recruited to promoters and coding regions of CWI Rlm1-dependent genes in response to stress. This phenomenon is dependent both on the activation of the MAPK and its kinase activity. Slt2 binding is also dependent on Rlm1 and SWI/SNF and SAGA complexes. During the initial steps of transcription, the catalytic activity of Slt2 on Rlm1 is critical for the binding of the activator to promoters in response to stress. In addition, Slt2 itself acts as a transactivator, as it is able to induce the transcription of CWI responsive genes when it is bound to promoters through the Rlm1 binding domain independently of its catalytic activity. Slt2 interacts with RNA Pol II in a Rlm1-dependent manner to provide further support to a role of this MAPK as an integral component of the transcriptional complexes under cell wall stress. Selective recruitment and progression of the complex Slt2-RNA Pol II from the promoters to the coding regions of Rlm1-dependent genes does not rely on Paf1, suggesting a different mechanism from that which is exerted by Slt2 on the Swi4/Swi6 (SBF)-regulated genes.

BrFLC5: a weak regulator of flowering time in Brassica rapa.

KEY MESSAGE: A splicing site mutation in BrFLC5, a non-syntenic paralogue of FLOWERING LOCUS C, was demonstrated to be related to flowering time variation in Brassica rapa. Flowering time regulation in Brassica rapa is more complex than in Arabidopsis, as there are multiple paralogues of flowering time genes in B. rapa. Brassica rapa contains four FLOWERING LOCUS C (FLC) genes, three of which are syntenic orthologues of AtFLC, while BrFLC5 is not. BrFLC1, BrFLC2, and BrFLC3 have been reported to be involved in flowering time regulation. However, BrFLC5 has thus far been deemed a pseudogene. We detected two alternative splicing patterns of BrFLC5 resulting from a nucleotide mutation (G/A) at the first nucleotide of intron 3 (named as Pi3+1(G/A)). Genotyping of BrFLC5Pi3 + 1(G/A) for 301 B. rapa accessions showed that this single nucleotide polymorphism was significantly related to flowering time variation (p < 0.001). In the collection, the frequency of the functional G allele (35.2%) was much lower than that of the nonfunctional A allele (59.1%); however, the frequency of the G allele was very high among the turnips (83.6%). An F2 population segregating at this locus was developed to analyze the genetic effect of BrFLC5. The result showed that the G allele individuals began to bolt two days later than the A allele individuals, indicating that BrFLC5 is a weak regulator of flowering time. BrFLC5 was expressed at the lowest level among the three analyzed BrFLCs. The late allele (G allele) was dominant to the early allele (A allele) at the BrFLC5 locus, which was in contrast to that of BrFLC1 and BrFLC2. This characteristic suggests that BrFLC5 would be more efficient for breeding premature bolting resistance in B. rapa.