Shorter Siliques Research Articles

The Arabidopsis ARID-HMG DNA-BINDING PROTEIN 15 modulates JA signaling by regulating MYC2 during pollen development.

The intricate process of male gametophyte development in flowering plants is regulated by jasmonic acid (JA) signaling. JA signaling initiates with the activation of the basic-helix-loop-helix (bHLH) transcription factor (TF), MYC2, leading to the expression of numerous JA-responsive genes during stamen development and pollen maturation. However, the regulation of JA signaling during different stages of male gametophyte development remains less understood. This study focuses on the characterization of the plant ARID-HMG DNA-BINDING PROTEIN 15 (AtHMGB15), and its role in pollen development in Arabidopsis (Arabidopsis thaliana). Phenotypic characterization of a T-DNA insertion line (athmgb15-4) revealed delayed bolting, shorter siliques, and reduced seed set in mutant plants compared to the wildtype. Additionally, AtHMGB15 deletion resulted in defective pollen morphology, delayed pollen germination, aberrant pollen tube growth, and a higher percentage of non-viable pollen grains. Molecular analysis indicated the down-regulation of JA biosynthesis and signaling genes in the athmgb15-4 mutant. Quantitative analysis demonstrated that jasmonic acid and its derivatives were approximately tenfold lower in athmgb15-4 flowers. Exogenous application of methyl jasmonate could restore pollen morphology and germination, suggesting that the low JA content in athmgb15-4 impaired JA signaling during pollen development. Furthermore, our study revealed that AtHMGB15 physically interacts with MYC2 to form a transcription activation complex. This complex promotes the transcription of key JA signaling genes, the R2R3-MYB TFs MYB21 and MYB24, during stamen and pollen development. Collectively, our findings highlight the role of AtHMGB15 as a positive regulator of the JA pathway, controlling the spatiotemporal expression of key regulators involved in Arabidopsis stamen and pollen development.

The Arabidopsis SR45 splicing factor bridges the splicing machinery and the exon-exon junction complex.

The Arabidopsis splicing factor serine/arginine-rich 45 (SR45) contributes to several biological processes. The sr45-1 loss-of-function mutant exhibits delayed root development, late flowering, unusual numbers of floral organs, shorter siliques with decreased seed sets, narrower leaves and petals, and altered metal distribution. SR45 bears a unique RNA recognition motif (RRM) flanked by one serine/arginine-rich (RS) domain on both sides. Here, we studied the function of each SR45 domains by examining their involvement in: (i) the spatial distribution of SR45; (ii) the establishment of a protein-protein interaction network including spliceosomal and exon-exon junction complex (EJC) components; and (iii) the RNA binding specificity. We report that the endogenous SR45 promoter is active during vegetative and reproductive growth, and that the SR45 protein localizes in the nucleus. We demonstrate that the C-terminal arginine/serine-rich domain is a determinant of nuclear localization. We show that the SR45 RRM domain specifically binds purine-rich RNA motifs via three residues (H101, H141, and Y143), and is also involved in protein-protein interactions. We further show that SR45 bridges both mRNA splicing and surveillance machineries as a partner of EJC core components and peripheral factors, which requires phosphoresidues probably phosphorylated by kinases from both the CLK and SRPK families. Our findings provide insights into the contribution of each SR45 domain to both spliceosome and EJC assemblies.

Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis.

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR-cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K+ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K+ homeostasis.

MiR319-Regulated TCP3 Modulates Silique Development Associated with Seed Shattering in Brassicaceae.

Seed shattering is an undesirable trait that leads to crop yield loss. Improving silique resistance to shattering is critical for grain and oil crops. In this study, we found that miR319-targeted TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCPs) inhibited the process of post-fertilized fruits (silique) elongation and dehiscence via regulation of FRUITFULL (FUL) expression in Arabidopsis thaliana and Brassica napus. AtMIR319a activation resulted in a longer silique with thickened and lignified replum, whereas overexpression of an miR319a-resistant version of AtTCP3 (mTCP3) led to a short silique with narrow and less lignified replum. Further genetic and expressional analysis suggested that FUL acted downstream of TCP3 to negatively regulate silique development. Moreover, hyper-activation of BnTCP3.A8, a B. napus homolog of AtTCP3, in rapeseed resulted in an enhanced silique resistance to shattering due to attenuated replum development. Taken together, our findings advance our knowledge of TCP-regulated silique development and provide a potential target for genetic manipulation to reduce silique shattering in Brassica crops.

Altered metal distribution in the sr45-1 Arabidopsis mutant causes developmental defects.

The plant serine/arginine-rich (SR) splicing factor SR45 plays important roles in several biological processes, such as splicing, DNA methylation, innate immunity, glucose regulation, and abscisic acid signaling. A homozygous Arabidopsis sr45-1 null mutant is viable, but exhibits diverse phenotypic alterations, including delayed root development, late flowering, shorter siliques with fewer seeds, narrower leaves and petals, and unusual numbers of floral organs. Here, we report that the sr45-1 mutant presents an unexpected constitutive iron deficiency phenotype characterized by altered metal distribution in the plant. RNA-Sequencing highlighted severe perturbations in metal homeostasis, the phenylpropanoid pathway, oxidative stress responses, and reproductive development. Ionomic quantification and histochemical staining revealed strong iron accumulation in the sr45-1 root tissues accompanied by iron starvation in aerial parts. Mis-splicing of several key iron homeostasis genes, including BTS, bHLH104, PYE, FRD3, and ZIF1, was observed in sr45-1 roots. We showed that some sr45-1 developmental abnormalities can be complemented by exogenous iron supply. Our findings provide new insight into the molecular mechanisms governing the phenotypes of the sr45-1 mutant.

BnaC7.ROT3, the causal gene of cqSL-C7, mediates silique length by affecting cell elongation in Brassica napus.

Siliques are a major carbohydrate source of energy for later seed development in rapeseed (Brassica napus). Thus, silique length has received great attention from breeders. We previously detected a novel quantitative trait locus cqSL-C7 that controls silique length in B. napus. Here, we further validated the cqSL-C7 locus and isolated its causal gene (BnaC7.ROT3) by map-based cloning. In 'Zhongshuang11' (parent line with long siliques), BnaC7.ROT3 encodes the potential cytochrome P450 monooxygenase CYP90C1, whereas in 'G120' (parent line with short siliques), a single nucleotide deletion in the fifth exon of BnaC7.ROT3 results in a loss-of-function truncated protein. Sub-cellular localization and expression pattern analysis revealed that BnaC7.ROT3 is a membrane-localized protein mainly expressed in leaves, flowers and siliques. Cytological observations showed that the cells in silique walls of BnaC7.ROT3-transformed positive plants were longer than those of transgene-negative plants in the background of 'G120', suggesting that BnaC7.ROT3 affects cell elongation. Haplotype analysis demonstrated that most alleles of BnaC7.ROT3 are favorable in B. napus germplasms, and its homologs may also be involved in silique length regulation. Our findings provide novel insights into the regulatory mechanisms of natural silique length variations and valuable genetic resources for the improvement of silique length in rapeseed.

FLA14 is required for pollen development and preventing premature pollen germination under high humidity in Arabidopsis

BackgroundAs an important subfamily of arabinogalactan proteins (AGPs), fasciclin-like AGPs (FLAs) contribute to various aspects of growth, development and adaptation, yet their function remains largely elusive. Despite the diversity of FLAs, only two members, Arabidopsis FLA3 and rice MTR1, are reported to be involved in sexual reproduction. In this study, another Arabidopsis FLA-encoding gene, FLA14, was identified, and its role was investigated.ResultsArabidopsis FLA14 was found to be a pollen grain-specific gene. Expression results from fusion with green fluorescent protein showed that FLA14 was localized along the cell membrane and in Hechtian strands. A loss-of-function mutant of FLA14 showed no discernible defects during male gametogenesis, but precocious pollen germination occurred inside the mature anthers under high moisture conditions. Overexpression of FLA14 caused 39.2% abnormal pollen grains with a shrunken and withered appearance, leading to largely reduced fertility with short mature siliques and lower seed set. Cytological and ultramicroscopic observation showed that ectopic expression of FLA14 caused disruption at the uninucleate stage, resulting in either collapsed pollen with absent intine or pollen of normal appearance but with a thickened intine.ConclusionsTaken together, our data suggest a role for FLA14 in pollen development and preventing premature pollen germination inside the anthers under high relative humidity in Arabidopsis.

Identification of transcription factors controlling cell wall invertase gene expression for reproductive development via bioinformatic and transgenic analyses.

Cell wall invertase (CWIN) hydrolyses sucrose into glucose and fructose in the extracellular matrix and plays crucial roles in assimilate partitioning and sugar signalling. However, the molecular regulators controlling CWIN gene transcription remain unknown. As the first step to address this issue, we performed bioinformatic and transgenic studies, which identified a cohort of transcription factors (TFs) modulating CWIN gene expression in Arabidopsis thaliana. Comprehensive bioinformatic analyses identified 18 TFs as putative regulators of the expression of AtCWIN2 and AtCWIN4 that are predominantly expressed in Arabidopsis reproductive organs. Among them, MYB21, ARF6, ARF8, AP3 and CRC were subsequently shown to be the most likely regulators of CWIN gene expression based on molecular characterization of the respective mutant of each candidate TF. More specifically, the obtained data indicate that ARF6, ARF8 and MYB21 regulate CWIN2 expression in the anthers and CWIN4 in nectaries, anthers and petals, whereas AP3 and CRC were determined primarily to regulate the transcriptional activity of CWIN4. TF-promoter interaction assays demonstrated that ARF6 and ARF8 directly control CWIN2 and CWIN4 transcription with AP3 activating CWIN4. The involvement of ARF8 in regulating CWIN4 expression was further supported by the finding that enhanced CWIN4 expression partially recovered the short silique phenotype displayed by the arf8-3 mutant. The identification of the five TFs regulating CWIN expression serves as a launching pad for future studies to dissect the upstream molecular network underpinning the transcription of CWINs and provides a new avenue, potentially, to engineer assimilate allocation and reproductive development for improving seed yield.

A novel Arabidopsis gene RGAT1 is required for GA-mediated tapetum and pollen development.

The phytohormone gibberellin (GA) is critical for anther development. RGA, a member of the DELLA family of proteins that are central GA signalling repressors, is a key regulator of male fertility in plants. However, the downstream genes in GA-RGA-mediated anther development remain to be characterised. We identified RGA Target 1 (RGAT1), a novel Arabidopsis gene, that functions as an important RGA-regulated target in pollen development. RGAT1 is predominantly expressed in the tapetum and microspores during anther stages 8-11, and can be directly activated by RGA and suppressed by GA in inflorescence apices. Both loss of function and gain of function of RGAT1 led to abnormal tapetum development, resulting in abortive pollen and short siliques. In RGAT1-knockdown and overexpression lines, pollen abortion occurred at stage 10. Loss of RGAT1 function induced the premature degeneration of tapetal cells with defective ER-derived tapetosomes, while RGAT1 overexpression delayed tapetum degeneration. TUNEL assay confirmed that RGAT1 participates in timely tapetal programmed cell death. Moreover, reducing RGAT1 expression partially rescued the tapetal developmental defects in GA-deficient ga1-3 mutant. Our findings revealed that RGAT1 is a direct target of RGA and plays an essential role in GA-mediated tapetum and pollen development.

Orchid Bsister gene PeMADS28 displays conserved function in ovule integument development

The ovules and egg cells are well developed to be fertilized at anthesis in many flowering plants. However, ovule development is triggered by pollination in most orchids. In this study, we characterized the function of a Bsister gene, named PeMADS28, isolated from Phalaenopsis equestris, the genome-sequenced orchid. Spatial and temporal expression analysis showed PeMADS28 predominantly expressed in ovules between 32 and 48 days after pollination, which synchronizes with integument development. Subcellular localization and protein–protein interaction analyses revealed that PeMADS28 could form a homodimer as well as heterodimers with D-class and E-class MADS-box proteins. In addition, ectopic expression of PeMADS28 in Arabidopsis thaliana induced small curled rosette leaves, short silique length and few seeds, similar to that with overexpression of other species’ Bsister genes in Arabidopsis. Furthermore, complementation test revealed that PeMADS28 could rescue the phenotype of the ABS/TT16 mutant. Together, these results indicate the conserved function of BsisterPeMADS28 associated with ovule integument development in orchid.

Genome-Wide DNA Methylation Analysis of Soybean Curled-Cotyledons Mutant and Functional Evaluation of a Homeodomain-Leucine Zipper (HD-Zip) I Gene GmHDZ20.

DNA methylation is a major, conserved epigenetic modification that influences many biological processes. Cotyledons are specialized tissues that provide nutrition for seedlings at the early developmental stage. To investigate the patterns of genomic DNA methylation of germinated cotyledons in soybean (Glycine max) and its effect on cotyledon development, we performed a genome-wide comparative analysis of DNA methylation between the soybean curled-cotyledons (cco) mutant, which has abnormal cotyledons, and its corresponding wild type (WT) by whole-genome bisulfite sequencing. The cco mutant was methylated at more sites but at a slightly lower level overall than the WT on the whole-genome level. A total of 46 CG-, 92 CHG-, and 9723 CHH- (H = A, C, or T) differentially methylated genes (DMGs) were identified in cotyledons. Notably, hypomethylated CHH-DMGs were enriched in the gene ontology term “sequence-specific DNA binding transcription factor activity.” We selected a DMG encoding a homeodomain-leucine zipper (HD-Zip) I subgroup transcription factor (GmHDZ20) for further functional characterization. GmHDZ20 localized to the nucleus and was highly expressed in leaf and cotyledon tissues. Constitutive expression of GmHDZ20 in Arabidopsis thaliana led to serrated rosette leaves, shorter siliques, and reduced seed number per silique. A yeast two-hybrid assay revealed that GmHDZ20 physically interacted with three proteins associated with multiple aspects of plant growth. Collectively, our results provide a comprehensive study of soybean DNA methylation in normal and aberrant cotyledons, which will be useful for the identification of specific DMGs that participate in cotyledon development, and also provide a foundation for future in-depth functional study of GmHDZ20 in soybean.

CRISPR-Cas9 multiplex genome editing of the hydroxyproline-O-galactosyltransferase gene family alters arabinogalactan-protein glycosylation and function in Arabidopsis

BackgroundArabinogalactan-proteins (AGPs) are a class of hydroxyproline-rich proteins (HRGPs) that are heavily glycosylated (> 90%) with type II arabinogalactans (AGs). AGPs are implicated in various plant growth and development processes including cell expansion, somatic embryogenesis, root and stem growth, salt tolerance, hormone signaling, male and female gametophyte development, and defense. To date, eight Hyp-O-galactosyltransferases (GALT2–6, HPGT1–3) have been identified; these enzymes are responsible for adding the first sugar, galactose, onto AGPs. Due to gene redundancy among the GALTs, single or double galt genetic knockout mutants are often not sufficient to fully reveal their biological functions.ResultsHere, we report the successful application of CRISPR-Cas9 gene editing/multiplexing technology to generate higher-order knockout mutants of five members of the GALT gene family (GALT2–6). AGPs analysis of higher-order galt mutants (galt2 galt5, galt3 galt4 galt6, and galt2 galt3 galt4 galt5 gal6) demonstrated significantly less glycosylated AGPs in rosette leaves, stems, and siliques compared to the corresponding wild-type organs. Monosaccharide composition analysis of AGPs isolated from rosette leaves revealed significant decreases in arabinose and galactose in all the higher-order galt mutants. Phenotypic analyses revealed that mutation of two or more GALT genes was able to overcome the growth inhibitory effect of β-D-Gal-Yariv reagent, which specifically binds to β-1,3-galactan backbones on AGPs. In addition, the galt2 galt3 galt4 galt5 gal6 mutant exhibited reduced overall growth, impaired root growth, abnormal pollen, shorter siliques, and reduced seed set. Reciprocal crossing experiments demonstrated that galt2 galt3 galt4 galt5 gal6 mutants had defects in the female gametophyte which were responsible for reduced seed set.ConclusionsOur CRISPR/Cas9 gene editing/multiplexing approach provides a simpler and faster way to generate higher-order mutants for functional characterization compared to conventional genetic crossing of T-DNA mutant lines. Higher-order galt mutants produced and characterized in this study provide insight into the relationship between sugar decorations and the various biological functions attributed to AGPs in plants.

Heterologous expression of chrysanthemum TOPLESS corepressor CmTPL1-1 alters meristem maintenance and organ development in Arabidopsis thaliana

TOPLESS (TPL)/TOPLESS-related (TPR) corepressors are important regulators of plant growth and development, but their functions in chrysanthemum (Chrysanthemum morifolium) are currently unclear. In this study, a chrysanthemum TPL/TPR family gene, designated CmTPL1-1, was characterized. This gene encodes an 1135-amino-acid polypeptide harboring a conserved N-terminal domain and two C-terminal WD40 domains. CmTPL1-1 showed no transcriptional activity in yeast, and a localization experiment indicated that it localized to the nuclei in onion epidermal cells. Transcript profiling established that the gene was most highly expressed in the stem apex. The heterologous expression of CmTPL1-1 in Arabidopsis thaliana produced a pleiotropic phenotype, including smaller leaves, shorter siliques, increased meristem number, asymmetrical petal distribution and reduced stamen number. In transgenic plants, four AtARFs were downregulated, while six AtIAAs and two AtGH3s were upregulated at the transcript level; moreover, the expression of three key class I KNOTTED-like homeobox (KNOX) genes was upregulated. In addition, by yeast two-hybrid screening of a chrysanthemum cDNA library, we found that CmTPL1-1 could interact with CmWOX4, CmLBD38 and CmLBD36, and these interactions were confirmed by bimolecular fluorescence complementation (BiFC) assays. Overall, we speculated that heterologous expression of CmTPL1-1 regulates plant growth and development by interacting with auxin signaling in Arabidopsis.

The Nuclear Localization of the DnaJ-Like Zinc Finger Domain-Containing Protein EDA3 Affects Seed Development in Arabidopsis thaliana.

The DnaJ-like zinc finger domain-containing proteins are involved in different aspects of plastid function and development. Some of these proteins were recently reported to have dual subcellular localization in the nucleus and plastids. One member of this family, PSA2 (AT2G34860), was found to localize to the thylakoid lumen and regulate the assembly of photosystem I (PSI). However, PSA2 was also annotated as Embryo sac Development Arrest 3 (EDA3) from the observation that its embryo sac development was arrested at the two-nuclear stage. In this study, we characterized the eda3 mutant, and demonstrated that, as compared with the wild-type (WT) plants, the mutant has shorter siliques, fewer siliques per plant, and fewer seeds per silique. Both aborted and undeveloped ovules were observed in siliques of the mutant. By immunoblot analysis, we found that, different from the chloroplast localization in mature leaves, EDA3 localizes in the nucleus in seeds. A nuclear localization signal was identified from the deduced amino acid sequence of EDA3, and also proved to be sufficient for directing its fusion peptide into the nucleus.

Targeted mutagenesis of EOD3 gene in Brassica napus L. regulates seed production.

Seed size and number are central to the evolutionary fitness of plants and are also crucial for seed production of crops. However, the molecular mechanisms of seed production control are poorly understood in Brassica crops. Here, we report the gene cloning, expression analysis, and functional characterization of the EOD3/CYP78A6 gene in rapeseed. BnaEOD3 has four copies located in two subgenomes, which exhibited a steady higher expression during seed development with differential expression among copies. The targeted mutations of BnaEOD3 gene were efficiently generated by stable transformation of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat) vector. These mutations were stably transmitted to T1 and T2 generations and a large collection of homozygous mutants with combined loss-of-function alleles across four BnaEOD3 copies were created for phenotyping. All mutant T1 lines had shorter siliques, smaller seeds, and an increased number of seeds per silique, in which the quadrable mutants showed the most significant changes in these traits. Consequently, the seed weight per plant in the quadrable mutants increased by 13.9% on average compared with that of wild type, indicating that these BnaEOD3 copies have redundant functions in seed development in rapeseed. The phenotypes of the different allelic combinations of BnaEOD3 copies also revealed gene functional differentiation among the two subgenomes. Cytological observations indicated that the BnaEOD3 could act maternally to promote cotyledon cell expansion and proliferation to regulate seed growth in rapeseed. Collectively, our findings reveal the quantitative involvement of the different BnaEOD3 copies function in seed development, but also provided valuable resources for rapeseed breeding programs.

Genome-wide analysis of CsWOX transcription factor gene family in cucumber (Cucumis sativus L.)

WUSCHEL-related homeobox (WOX) transcription factors are plant-specific members that characterized by the presence of a homeodomain. It has been shown that WOX members regulate several aspects of plant development, but the biological functions of this CsWOX gene family remain largely unknown in cucumber (Cucumis sativus L.). In this study, we identified and characterized 11 putative CsWOX genes in cucumber, which are also divided into three major clades (e.g., the Ancient clade, the Intermediate clade and the WUS clade). Expression pattern analysis revealed tissue-specific expression patterns of CsWOX genes, including that CsWOX9 is mainly expressed in developing fruit and also has lower expression in tip and axillary bud, which was further confirmed by in situ hybridization assay. Moreover, overexpression of CsWOX9 in Arabidopsis led to increased branches and rosette leaves, and shorter siliques. Together, these results indicated that CsWOX members may regulate cucumber growth and development.

The beta Subunit of Nascent Polypeptide Associated Complex Plays A Role in Flowers and Siliques Development of Arabidopsis thaliana.

The nascent polypeptide-associated (NAC) complex was described in yeast as a heterodimer composed of two subunits, α and β, and was shown to bind to the nascent polypeptides newly emerging from the ribosomes. NAC function was widely described in yeast and several information are also available about its role in plants. The knock down of individual NAC subunit(s) led usually to a higher sensitivity to stress. In Arabidopsis thaliana genome, there are five genes encoding NACα subunit, and two genes encoding NACβ. Double homozygous mutant in both genes coding for NACβ was acquired, which showed a delayed development compared to the wild type, had abnormal number of flower organs, shorter siliques and greatly reduced seed set. Both NACβ genes were characterized in more detail—the phenotype of the double homozygous mutant was complemented by a functional NACβ copy. Then, both NACβ genes were localized to nuclei and cytoplasm and their promoters were active in many organs (leaves, cauline leaves, flowers, pollen grains, and siliques together with seeds). Since flowers were the most affected organs by nacβ mutation, the flower buds’ transcriptome was identified by RNA sequencing, and their proteome by gel-free approach. The differential expression analyses of transcriptomic and proteomic datasets suggest the involvement of NACβ subunits in stress responses, male gametophyte development, and photosynthesis.

Ectopic expression of IiSHP2 from Isatis indigotica Fortune, a PLE-lineage MADS-box gene, influences leaf, floral organ and silique morphology in Arabidopsis thaliana.

In order to ascertain the regulatory mechanism of fruit development in Isatis indigotica Fortune, the complementary DNA (cDNA) sequence of the SHATTERPROOF 2 (SHP2) orthologous gene was identified by Rapid Amplification of cDNA Ends technology and the corresponding gene was named IiSHP2. The expression pattern of IiSHP2 was determined by quantitative reverse transcription-polymerase chain reaction and wild-type Col-0 Arabidopsis plants were transformed with the IiSHP2 gene using Agrobacterium tumefaciens and the floral-dip method. Expression analyses indicated that IiSHP2 was highly expressed in flowers, silicles and seeds. Compared to wild-type plants, IiSHP2 transgenic lines bolted earlier. Detailed phenotypic observations showed that the size of the rosette and cauline leaves in transgenic lines was reduced and the cauline leaves of the transgenic lines were incurved and displayed a funnel-like shape. During the reproductive growth stage, IiSHP2 transgenic plants produced shortened sepals and the flower buds were not encapsulated completely. Moreover, the petals of the transgenic lines were converted into stamineous tissues, accompanied by exposed stamens, short malformed siliques and wrinkled valves, indicating a severe decline in fertility. These experimental conclusions are valuable as a reference for the breeding of medicinal plants.

A gain-of-function mutation in Brassinosteroid-insensitive 2 alters Arabidopsis floral organ development by altering auxin levels.

Auxin can alter the fertility of bin2-1 plants and depends on the expression of SHY2. Brassinosteroids (BRs) play important roles in plant growth and developmental processes. By systematically evaluating the phenotypes of BR biosynthesis and BR signaling mutants, researchers have reported that BRs positively regulate floral development. In this study, we found that brassinosteroid-insensitive 2 (bin2-1) and short-hypocotyl 2 (shy2-2) mutants exhibited significantly reduced fertility. These mutants had short inflorescences, decreased floral organ length (short petals, stamens, carpels, and stigmas), and short siliques. Exogenous auxin applications could partially rescue the shortened length of the floral organs and siliques of the bin2-1 mutants. Additional experiments revealed that a lack of SHY2 activity increased the fertility of the bin2-1 mutants. A search for downstream affected genes revealed that auxin influences the expression of ARFs and PINs in the bin2-1 mutants, suggesting that auxin plays a major role in the regulation of bin2-1 plant fertility. Thus, BIN2 plays a role in fertility by affecting auxin levels, mainly by altering the expression of SHY2.

Ectopic Expression of Litsea cubeba LcMADS20 Modifies Silique Architecture.

Litsea cubeba (Lour.) Pers. (mountain pepper, Lauraceae) is an important woody essential oil crop that produces fragrant oils in its fruits, especially in its peels. Identification of genes involved in the regulation of fruits and peel architecture is of economic significance for L. cubeba industry. It has been well known that the MADS-box genes are essential transcription factors that control flowers and fruits development. Here, we obtained 33 MADS-box genes first from the RNA-seq data in L. cubeba, and 27 of these genes were of the MIKC-type. LcMADS20, an AGAMOUS-like gene, was highly expressed in the developing stages of fruits, particularly at 85 days after full bloom. The ectopic expression of LcMADS20 in Arabidopsis resulted in not only curved leaves, early flowering and early full-opened inflorescences, but also shorter siliques and decreased percentage of peel thickness. Moreover, in the LcMADS20 transgenic Arabidopsis, the expression modes of several intrinsic ABC model class genes were influenced, among which the expression of FUL was significantly reduced and AP3, AG, and STK were significantly increased. This study systematically analyzed the MADS-box genes in L. cubeba at the transcriptional level and showed that LcMADS20 plays important roles in the regulation of fruit architecture.