The chloroplast pentatricopeptide repeat protein RCN22 regulates tiller number in rice by affecting sugar levels via the TB1-RCN22-RbcL module.

As an important yield component, the rice tiller number controls panicle number and determines grain yield. The regulation of rice tiller number by chloroplast pentatricopeptide repeat (PPR) proteins has not been reported. Here, we report a rice reduced culm number22 (rcn22) mutant which produces few tillers due to suppressed tiller bud elongation. Map-based cloning revealed that RCN22 encodes a chloroplast-localized P-type PPR protein. We found that RCN22 specifically binds to the 5'-UTR of RbcL mRNA (encoding the large subunit of Rubisco) and enhances its stability. The reduced RbcL mRNA abundance in rcn22 led to a lower photosynthetic rate and decreased sugar levels. Consequently, transcript levels of DWARF3 (D3) and TEOSINTE BRANCHED1 (TB1) (encoding negative regulators of tiller bud elongation) increased, whereas protein levels of a positive regulator DWARF53 (D53) decreased. Furthermore, high concentrations of sucrose could rescue the tiller bud growth defect of the rcn22 mutant. On the other hand, TB1 directly binds to the RCN22 promoter and downregulates its expression. The tb1/rcn22 double mutant showed a tillering phenotype similar to rcn22. Our results suggest that the TB1-RCN22-RbcL module plays a vital role in rice tiller bud elongation by affecting sugar levels.

The lncRNA1-miR6288b-3p-PpTCP4-PpD2 module regulates peach branch number by affecting brassinosteroid biosynthesis.

Branch number is one of the most important agronomic traits of fruit trees such as peach. Little is known about how LncRNA and/or miRNA modules regulate branching through transcription factors. Here, we used molecular and genetic tools to clarify the molecular mechanisms underlying brassinosteroid (BR) altering plant branching. We found that the number of sylleptic branch and BR content in pillar peach ('Zhaoshouhong') was lower than those of standard type ('Okubo'), and exogenous BR application could significantly promote branching. PpTCP4 expressed great differentially comparing 'Zhaoshouhong' with 'Okubo'. PpTCP4 could directly bind to DWARF2 (PpD2) and inhibited its expression. PpD2 was the only one differentially expressed key gene in the path of BR biosynthesis. At the same time, PpTCP4 was identified as a target of miR6288b-3p. LncRNA1 could act as the endogenous target mimic of miR6288b-3p and repress expression of miR6288b-3p. Three deletions and five SNP sites of lncRNA1 promoter were found in 'Zhaoshouhong', which was an important cause of different mRNA level of PpTCP4 and BR content. Moreover, overexpressed PpTCP4 significantly inhibited branching. A novel mechanism in which the lncRNA1-miR6288b-3p-PpTCP4-PpD2 module regulates peach branching number was proposed.

Flowering time genes branching out.

Plants are sessile by nature, and as such they have evolved to sense changes in seasonality and their surrounding environment, and adapt to these changes. One prime example of this is the regulation of flowering time in angiosperms, which is precisely timed by the coordinated action of two proteins: FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL1). Both of these regulators are members of the PHOSPHATIDYLETHANOLAMINE BINDING PROTEIN (PEBP) family of proteins. These regulatory proteins do not interact with DNA themselves, but instead interact with transcriptional regulators, such as FLOWERING LOCUS D (FD). FT and TFL1 were initially identified as key regulators of flowering time, acting through binding with FD; however, PEBP family members are also involved in shaping plant architecture and development. In addition, PEBPs can interact with TCP transcriptional regulators, such as TEOSINTE BRANCHED 1 (TB1), a well-known regulator of plant architecture, and key domestication-related genes in many crops. Here, we review the role of PEBPs in flowering time, plant architecture, and development. As these are also key yield-related traits, we highlight examples from the model plant Arabidopsis as well as important food and feed crops such as, rice, barley, wheat, tomato, and potato.

In-frame mutation in rice TEOSINTE BRANCHED1 (OsTB1) improves productivity under phosphorus deficiency.

Tillering is an important trait in rice productivity. We introduced mutations into the coding region of rice TEOSINTE BRANCHED1 (OsTB1), which is a negative regulator of tillering, using CRISPR/Cas9. The frameshift mutants exhibited substantially enhanced tillering and produced 3.5 times more panicles than the non-mutated plants at maturity. This enhanced tillering resulted in increased spikelet number; however, grain yields did not increase due to substantially reduced filled grain rate and 1,000-grain weight. In contrast, in-frame mutations in OsTB1 had the effect of slightly increasing tiller numbers, and the in-frame mutants had 40% more panicles than non-mutated plants. The grain yield of in-frame mutants also did not increase on nutrient-rich soil; however, under phosphorus-deficient conditions, where tillering is constrained, the in-frame mutants gave a significantly higher grain yield than non-mutated plants due to higher spikelet number and maintained filled grain rate. Rice grassy tiller1 (OsGT1)/OsHox12, which is directly regulated by OsTB1 to suppress tillering, was moderately down-regulated in in-frame mutants, suggesting that OsTB1 with the in-frame mutation shows partial function of intact OsTB1 in regulating OsGT1/OsHox12. We propose that mildly enhanced tillering by in-frame mutation of OsTB1 can improve grain yield under low phosphorus conditions.

PIL transcription factors directly interact with SPLs and repress tillering/branching in plants.

Tillering is an important parameter of plant architecture in cereal crops. In this study, we identified the PHYTOCHROME-INTERACTING FACTOR-LIKE (PIL) family transcription factors as new repressors of tillering in cereal crops. Using biochemical and genetic approaches, we explore the roles of TaPIL1 in regulating wheat plant architecture. We found that the PIL protein TaPIL1 controls tiller number in wheat. Overexpression of TaPIL1 reduces wheat tiller number; additionally, overexpression of TaPIL1-SUPERMAN repression domain increases wheat tiller number. Furthermore, we show that TaPIL1 activates the transcriptional expression of wheat TEOSINTE BRANCHED1 (TaTB1); moreover, TaPIL1 physically interacts with wheat SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (TaSPL)3/17, which are activators of TaTB1 transcription. In rice, overexpression and loss-of-function mutations of OsPIL11 reduce or increase tiller number by regulating the expression of OsTB1. In Arabidopsis, we demonstrate that PHYTOCHROME-INTERACTING FACTOR 4 interacts with SPL9 to inhibit shoot branching. This study reveals that PIL family transcription factors directly interact with SPLs and play an important role in repressing tillering/branching in plants.

Expression and Function Studies of CYC/TB1-Like Genes in the Asymmetric Flower Canna (Cannaceae, Zingiberales).

The asymmetric flower, lacking any plane of symmetry, is rare among angiosperms. Canna indica L. has conspicuously asymmetric flowers resulting from the presence of a half-fertile stamen, while the other androecial members develop as petaloid staminodes or abort early during development. The molecular basis of the asymmetric distribution of fertility and petaloidy in the androecial whorls remains unknown. Ontogenetic studies have shown that Canna flowers are borne on monochasial (cincinnus) partial florescences within a racemose inflorescence, with floral asymmetry likely corresponding to the inflorescence architecture. Given the hypothesized role of CYC/TB1 genes in establishing floral symmetry in response to the influence of the underlying inflorescence architecture, the spatiotemporal expression patterns of three Canna CYC/TB1 homologs (CiTBL1a, CiTBL1b-1, and CiTBL1b-2) were analyzed during inflorescence and floral development using RNA in situ hybridization and qRT-PCR. In the young inflorescence, both CiTBL1a and CiTBL1b-1 were found to be expressed in the bracts and at the base of the lateral florescence branches, whereas transcripts of CiTBL1b-2 were mainly detected in flower primordia and inflorescence primordia. During early flower development, expression of CiTBL1a and CiTBL1b-1 were both restricted to the developing sepals and petals. In later flower development, expression of CiTBL1a was reduced to a very low level while CiTBL1b-1 was detected with extremely high expression levels in the petaloid androecial structures including the petaloid staminodes, the labellum, and the petaloid appendage of the fertile stamen. In contrast, expression of CiTBL1b-2 was strongest in the fertile stamen throughout flower development, from early initiation of the stamen primordium to maturity of the ½ anther. Heterologous overexpression of CiTBL genes in Arabidopsis led to dwarf plants with smaller petals and fewer stamens, and altered the symmetry of mature flowers. These data provide evidence for the involvement of CYC/TB1 homologs in the development of the asymmetric Cannaceae flower.

Functional Analysis of the teosinte branched 1 Gene in the Tetraploid Switchgrass (Panicum virgatum L.) by CRISPR/Cas9-Directed Mutagenesis.

Tillering is an important biomass yield component trait in switchgrass (Panicum virgatum L.). Teosinte branched 1 (tb1)/Branched 1 (BRC1) gene is a known regulator for tillering/branching in several plant species; however, its role on tillering in switchgrass remains unknown. Here, we report physiological and molecular characterization of mutants created by CRISPR/Cas9. We successfully obtained nonchimeric Pvtb1a and Pvtb1b mutants from chimeric T0 mutants using nodal culture. The biallelic Pvtb1a-Pvtb1b mutant plants produced significantly more tillers and higher fresh weight biomass than the wild-type plants. The increased tiller number in the mutant plants resulted primarily from hastened outgrowth of lower axillary buds. Increased tillers were also observed in transgene-free BC1 monoallelic mutants for either Pvtb1a-Pvtb1b or Pvtb1b gene alone, suggesting Pvtb1 genes act in a dosage-dependent manner. Transcriptome analysis showed 831 genes were differentially expressed in the Pvtb1a-Pvtb1b double knockdown mutant. Gene Ontology analysis revealed downregulation of Pvtb1 genes affected multiple biological processes, including transcription, flower development, cell differentiation, and stress/defense responses in edited plants. This study demonstrates that Pvtb1 genes play a pivotal role in tiller production as a negative regulator in switchgrass and provides opportunities for further research aiming to elucidate the molecular pathway regulating tillering in switchgrass.

TEOSINTE BRANCHED1 regulates height and stem internode length in bread wheat.

Regulation of plant height and stem elongation has contributed significantly to improvement of cereal productivity by reducing lodging and improving distribution of assimilates to the inflorescence and grain. In wheat, genetic control of height has been largely contributed by the Reduced height-1 alleles that confer gibberellin insensitivity; the beneficial effects of these alleles are associated with less favourable effects involving seedling emergence, grain quality, and inflorescence architecture that have driven new research investigating genetic variation of stem growth. Here, we show that TEOSINTE BRANCHED1 (TB1) regulates height of wheat, with TB1 being expressed at low levels in nodes of the main culm prior to elongation, and increased dosage of TB1 restricting elongation of stem internodes. The effect of TB1 on stem growth is not accompanied by poor seedling emergence, as transgenic lines with increased activity of TB1 form longer coleoptiles than null transgenic controls. Analysis of height in a multiparent mapping population also showed that allelic variation for TB1 on the B genome influences height, with plants containing the variant TB-B1b allele being taller than those with the wild-type TB-B1a allele. Our results show that TB1 restricts height and stem elongation in wheat, suggesting that variant alleles that alter the expression or function of TB1 could be used as a new source of genetic diversity for optimizing architecture of wheat in breeding programmes.

Transcription factor TCP20 regulates peach bud endodormancy by inhibiting DAM5/DAM6 and interacting with ABF2.

The dormancy-associated MADS-box (DAM) genes PpDAM5 and PpDAM6 have been shown to play important roles in bud endodormancy; however, their molecular regulatory mechanism in peach is unclear. In this study, by use of yeast one-hybrid screening, we isolated a TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR transcription factor, PpTCP20, in the peach cultivar 'Zhongyou 4' (Prunus persica var. nectarina). The protein was localized in the nucleus and was capable of forming a homodimer. Electrophoretic mobility shift assays demonstrated that PpTCP20 binds to a GCCCR element in the promoters of PpDAM5 and PpDAM6, and transient dual luciferase experiments showed that PpTCP20 inhibited the expression of PpDAM5 and PpDAM6 as the period of the release of flower bud endodormancy approached. In addition, PpTCP20 interacted with PpABF2 to form heterodimers to regulate bud endodormancy, and the content of abscisic acid decreased with the release of endodormancy. PpTCP20 also inhibited expression of PpABF2 to regulate endodormancy. Taken together, our results suggest that PpTCP20 regulates peach flower bud endodormancy by negatively regulating the expression of PpDAM5 and PpDAM6, and by interacting with PpABF2, thus revealing a novel regulatory mechanism in a perennial deciduous tree.

The MPK8-TCP14 pathway promotes seed germination in Arabidopsis.

The accurate control of dormancy release and germination is critical for successful plantlet establishment. Investigations in cereals hypothesized a crucial role for specific MAP kinase (MPK) pathways in promoting dormancy release, although the identity of the MPK involved and the downstream events remain unclear. In this work, we characterized mutants for Arabidopsis thaliana MAP kinase 8 (MPK8). Mpk8 seeds presented a deeper dormancy than wild-type (WT) at harvest that was less efficiently alleviated by after-ripening and gibberellic acid treatment. We identified Teosinte Branched1/Cycloidea/Proliferating cell factor 14 (TCP14), a transcription factor regulating germination, as a partner of MPK8. Mpk8 tcp14 double-mutant seeds presented a deeper dormancy at harvest than WT and mpk8, but similar to that of tcp14 seeds. MPK8 interacted with TCP14 in the nucleus in vivo and phosphorylated TCP14 in vitro. Furthermore, MPK8 enhanced TCP14 transcriptional activity when co-expressed in tobacco leaves. Nevertheless, the stimulation of TCP14 transcriptional activity by MPK8 could occur independently of TCP14 phosphorylation. The comparison of WT, mpk8 and tcp14 transcriptomes evidenced that whereas no effect was observed in dry seeds, mpk8 and tcp14 mutants presented dramatic transcriptomic alterations after imbibition with a sustained expression of genes related to seed maturation. Moreover, both mutants exhibited repression of genes involved in cell wall remodeling and cell cycle G1/S transition. As a whole, this study unraveled a role for MPK8 in promoting seed germination, and suggested that its interaction with TCP14 was critical for regulating key processes required for germination completion.

Molecular cloning and sequence variance analysis of the TEOSINTE BRANCHED1 (TB1) gene in bermudagrass [Cynodon dactylon (L.) Pers].

TEOSINTE BRANCHED1 (TB1) encodes a TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL FACTOR (TCP) transcription factor that represses axillary bud outgrowth and lateral branch formation in plants. Previous studies have elucidated the essential tillering regulatory roles of TB1 in many grasses, including maize and rice; however, the functions of TB1 in turf grasses remain unclear. In this study, we cloned the CdTB1 gene from bermudagrass, an important turfgrass species, and characterized the transactivation function of the CdTB1 protein. Sequencing the CdTB1 gene locus in a mini-core germplasm collection of Chinese bermudagrasses led to the successful identification of 66 SNP and 2 indel mutations in the protein-coding region as well as 28 SNP and 11 indel mutations in the promoter region. Interestingly, mutations in the C-terminal transactivation domain of the CdTB1 protein had no significant influence on the transactivation activity, whereas a novel 335-bp insertion mutation located in the promoter region could significantly increase the expression of the CdTB1 gene. Furthermore, wild accessions of bermudagrass harboring the novel insertion mutation were found to have significantly reduced tillers compared with other accessions, suggesting a negative correlation between the mutation and tillering. The results of this study not only expanded our knowledge of TB1 gene expression regulation but also provided possible molecular markers to breed cultivars of turf and forage grasses with specific architectural features.

A Gene for Genetic Background in Zea mays: Fine-Mapping enhancer of teosinte branched1.2 to a YABBY Class Transcription Factor.

The effects of an allelic substitution at a gene often depend critically on genetic background, i.e., the genotypes at other genes in the genome. During the domestication of maize from its wild ancestor (teosinte), an allelic substitution at teosinte branched (tb1) caused changes in both plant and ear architecture. The effects of tb1 on phenotype were shown to depend on multiple background loci, including one called enhancer of tb1.2 (etb1.2). We mapped etb1.2 to a YABBY class transcription factor (ZmYAB2.1) and showed that the maize alleles of ZmYAB2.1 are either expressed at a lower level than teosinte alleles or disrupted by insertions in the sequences. tb1 and etb1.2 interact epistatically to control the length of internodes within the maize ear, which affects how densely the kernels are packed on the ear. The interaction effect is also observed at the level of gene expression, with tb1 acting as a repressor of ZmYAB2.1 expression. Curiously, ZmYAB2.1 was previously identified as a candidate gene for another domestication trait in maize, nonshattering ears. Consistent with this proposed role, ZmYAB2.1 is expressed in a narrow band of cells in immature ears that appears to represent a vestigial abscission (shattering) zone. Expression in this band of cells may also underlie the effect on internode elongation. The identification of ZmYAB2.1 as a background factor interacting with tb1 is a first step toward a gene-level understanding of how tb1 and the background within which it works evolved in concert during maize domestication.

Tillering in the sugary1 sweet corn is maintained by overriding the teosinte branched1 repressive signal.

The evolution of apical dominance in maize during domestication from teosinte is associated with higher expression from the teosinte branched1 (tb1) gene that inhibits tiller bud outgrowth. Unlike many standard maize varieties, the sweet corn inbred P39 that carries a mutation in a starch biosynthesis gene sugary1 produces multiple tillers and providing an opportunity to explore the diversification of the tb1 signal in maize. Through gene expression analysis, we show that tiller buds in P39 continue to grow by overriding the high expression level of tb1 that arrests bud outgrowth in maize inbred B73. In addition, we demonstrate that while B73 is largely non-responsive to shade, both P39 and teosinte respond through tb1-independent and tb1-dependent molecular mechanisms, respectively, leading to inhibition of tiller bud outgrowth.

Natural variation in teosinte at the domestication locus teosinte branched1 (tb1).

The teosinte branched1(tb1) gene is a major QTL controlling branching differences between maize and its wild progenitor, teosinte. The insertion of a transposable element (Hopscotch) upstream of tb1 is known to enhance the gene's expression, causing reduced tillering in maize. Observations of the maize tb1 allele in teosinte and estimates of an insertion age of the Hopscotch that predates domestication led us to investigate its prevalence and potential role in teosinte. We assessed the prevalence of the Hopscotch element across an Americas-wide sample of 837 maize and teosinte individuals using a co-dominant PCR assay. Additionally, we calculated population genetic summaries using sequence data from a subset of individuals from four teosinte populations and collected phenotypic data using seed from a single teosinte population where Hopscotch was found segregating at high frequency. Genotyping results indicate the Hopscotch element is found in a number of teosinte populations and linkage disequilibrium near tb1 does not support recent introgression from maize. Population genetic signatures are consistent with selection on the tb1 locus, revealing a potential ecological role, but a greenhouse experiment does not detect a strong association between the Hopscotch and tillering in teosinte. Our findings suggest the role of Hopscotch differs between maize and teosinte. Future work should assess tb1 expression levels in teosinte with and without the Hopscotch and more comprehensively phenotype teosinte to assess the ecological significance of the Hopscotch insertion and, more broadly, the tb1 locus in teosinte.

CINCINNATA in Antirrhinum majus directly modulates genes involved in cytokinin and auxin signaling.

Mutations in the CINCINNATA (CIN) gene in Antirrhinum majus and its orthologs in Arabidopsis result in crinkly leaves as a result of excess growth towards the leaf margin. CIN homologs code for TCP (TEOSINTE-BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR 1 AND 2) transcription factors and are expressed in a broad zone in a growing leaf distal to the proliferation zone where they accelerate cell maturation. Although a few TCP targets are known, the functional basis of CIN-mediated leaf morphogenesis remains unclear. We compared the global transcription profiles of wild-type and the cin mutant of A. majus to identify the targets of CIN. We cloned and studied the direct targets using RNA in situ hybridization, DNA-protein interaction, chromatin immunoprecipitation and reporter gene analysis. Many of the genes involved in the auxin and cytokinin signaling pathways showed altered expression in the cin mutant. Further, we showed that CIN binds to genomic regions and directly promotes the transcription of a cytokinin receptor homolog HISTIDINE KINASE 4 (AmHK4) and an IAA3/SHY2 (INDOLE-3-ACETIC ACID INDUCIBLE 3/SHORT HYPOCOTYL 2) homolog in A. majus. Our results suggest that CIN limits excess cell proliferation and maintains the flatness of the leaf surface by directly modulating the hormone pathways involved in patterning cell proliferation and differentiation during leaf growth.

Tillering and panicle branching genes in rice.

Rice (Oryza sativa L.) is one of the most important staple food crops in the world, and rice tillering and panicle branching are important traits determining grain yield. Since the gene MONOCULM 1 (MOC 1) was first characterized as a key regulator in controlling rice tillering and branching, great progress has been achieved in identifying important genes associated with grain yield, elucidating the genetic basis of yield-related traits. Some of these important genes were shown to be applicable for molecular breeding of high-yielding rice. This review focuses on recent advances, with emphasis on rice tillering and panicle branching genes, and their regulatory networks.

MicroRNA expression analysis of rosette and folding leaves in Chinese cabbage using high-throughput Solexa sequencing.

In this study, a global analysis of miRNA expression from rosette leaves (RLs) and folding leaves (FLs) of Chinese cabbage (Brassica rapa L. ssp. pekinensis) was conducted using high-throughput Solexa sequencing. In total, over 12 million clean reads were obtained from each library. Sequence analysis identified 64 conserved miRNA families in each leaf type and 104 and 95 novel miRNAs from RLs and FLs, respectively. Among these, 61 conserved miRNAs and 61 novel miRNAs were detected in both types of leaves. Furthermore, six conserved and 21 novel miRNAs were differentially expressed between the two libraries. Target gene annotation suggested that these differentially expressed miRNAs targeted transcription factors, F-box proteins, auxin and Ca(2+) signaling pathway proteins, protein kinases and other proteins that may function in governing leafy head formation. This study advanced our understanding of the important roles of miRNAs in regulating leafy head development in Chinese cabbage.