Dazhong Zhao

Signaling of cell fate determination by the TPD1 small protein and EMS1 receptor kinase.

Sexual reproduction requires the specification of cells with distinct fates in plants and animals. The EMS1 (also known as EXS) leucine-rich repeat receptor-like kinase (LRR-RLK) and TPD1 small protein play key roles in regulating somatic and reproductive cell fate determination in Arabidopsis anthers. Here, we show that ectopic expression of TPD1 causes abnormal differentiation of somatic and reproductive cells in anthers. In addition, ectopic TPD1 activity requires functional EMS1. Yeast two-hybrid, pull-down, and coimmunoprecipitation analyses further demonstrate that TPD1 interacts with EMS1 in vitro and in vivo. Moreover, TPD1 induces EMS1 phosphorylation in planta. Thus, our results suggest that TPD1 serves as a ligand for the EMS1 receptor kinase to signal cell fate determination during plant sexual reproduction.

Members of the Arabidopsis-SKP1-like Gene Family Exhibit a Variety of Expression Patterns and May Play Diverse Roles in Arabidopsis

Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, we tested for expression of 20 ASK genes using reverse transcription-polymerase chain reaction experiments. Also, we examined the expression patterns of 11 ASK genes by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, we selectively suppressed the expression of a few ASK genes using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibited similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes.

Regulation of Flower Development in Arabidopsis by SCF Complexes

SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.

GmFT2a, a soybean homolog of FLOWERING LOCUS T, is involved in flowering transition and maintenance.

Background Flowering reversion can be induced in soybean (Glycine max L. Merr.), a typical short-day (SD) dicot, by switching from SD to long-day (LD) photoperiods. This process may involve florigen, putatively encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana. However, little is known about the potential function of soybean FT homologs in flowering reversion. Methods A photoperiod-responsive FT homologue GmFT (renamed as GmFT2a hereafter) was cloned from the photoperiod-sensitive cultivar Zigongdongdou. GmFT2a gene expression under different photoperiods was analyzed by real-time quantitative PCR. In situ hybridization showed direct evidence for its expression during flowering-related processes. GmFT2a was shown to promote flowering using transgenic studies in Arabidopsis and soybean. The effects of photoperiod and temperature on GmFT2a expression were also analyzed in two cultivars with different photoperiod-sensitivities. Results GmFT2a expression is regulated by photoperiod. Analyses of GmFT2a transcripts revealed a strong correlation between GmFT2a expression and flowering maintenance. GmFT2a transcripts were observed continuously within the vascular tissue up to the shoot apex during flowering. By contrast, transcripts decreased to undetectable levels during flowering reversion. In grafting experiments, the early-flowering, photoperiod-insensitive stock Heihe27 promotes the appearance of GmFT2a transcripts in the shoot apex of scion Zigongdongdou under noninductive LD conditions. The photothermal effects of GmFT2a expression diversity in cultivars with different photoperiod-sensitivities and a hypothesis is proposed. Conclusion GmFT2a expression is associated with flowering induction and maintenance. Therefore, GmFT2a is a potential target gene for soybean breeding, with the aim of increasing geographic adaptation of this crop.

The SPOROCYTELESS/NOZZLE Gene Is Involved in Controlling Stamen Identity in Arabidopsis

The stamen, which consists of an anther and a filament, is the male reproductive organ in a flower. The specification of stamen identity in Arabidopsis (Arabidopsis thaliana) is controlled by a combination of the B genes APETALA3 (AP3) and PISTILLATA, the C gene AGAMOUS (AG), and the E genes SEPALLATA1 (SEP1) to SEP4. The “floral organ-building” gene SPOROCYTELESS/NOZZLE (SPL/NZZ) plays a central role in regulating anther cell differentiation. However, much less is known about how “floral organ identity” and floral organ-building genes interact to control floral organ development. In this study, we report that ectopic expression of SPL/NZZ not only affects flower development in the wild-type background but also leads to the transformation of petal-like organs into stamen-like organs in flowers of ap2-1, a weak ap2 mutant allele. Moreover, our loss-of-function analysis indicates that the spl/nzz mutant enhances the phenotype of the ag weak allele ag-4. Furthermore, ectopic expression and overexpression of SPL/NZZ altered expression of AG, SEP3, and AP2 in rosette leaves and flowers, while ectopic expression of SPL/NZZ resulted in ectopic expression of AG and SEP3 in the outer whorls of flowers. Our results indicate that the SPL/NZZ gene is engaged in controlling stamen identity via interacting with genes required for stamen identity in Arabidopsis.

Deregulation of the OsmiR160 Target Gene OsARF18 Causes Growth and Developmental Defects with an Alteration of Auxin Signaling in Rice

MicroRNAs (miRNAs) control gene expression as key negative regulators at the post-transcriptional level. MiR160 plays a pivotal role in Arabidopsis growth and development through repressing expression of its target AUXIN RESPONSE FACTOR (ARF) genes; however, the function of miR160 in monocots remains elusive. In this study, we found that the mature rice miR160 (OsmiR160) was mainly derived from OsMIR160a and OsMIR160b genes. Among four potential OsmiR160 target OsARF genes, the OsARF18 transcript was cleaved at the OsmiR160 target site. Rice transgenic plants (named mOsARF18) expressing an OsmiR160-resistant version of OsARF18 exhibited pleiotropic defects in growth and development, including dwarf stature, rolled leaves, and small seeds. mOsARF18 leaves were abnormal in bulliform cell differentiation and epidermal cell division. Starch accumulation in mOsARF18 seeds was also reduced. Moreover, auxin induced expression of OsMIR160a, OsMIR160b, and OsARF18, whereas expression of OsMIR160a and OsMIR160b as well as genes involved in auxin signaling was altered in mOsARF18 plants. Our results show that negative regulation of OsARF18 expression by OsmiR160 is critical for rice growth and development via affecting auxin signaling, which will advance future studies on the molecular mechanism by which miR160 fine-tunes auxin signaling in plants.

A common miRNA160‐based mechanism regulates ovary patterning, floral organ abscission and lamina outgrowth in tomato

Plant microRNAs play vital roles in auxin signaling via the negative regulation of auxin response factors (ARFs). Studies have shown that targeting of ARF10/16/17 by miR160 is indispensable for various aspects of development, but its functions in the model crop tomato (Solanum lycopersicum) are unknown. Here we knocked down miR160 (sly-miR160) using a short tandem target mimic (STTM160), and investigated its roles in tomato development. Northern blot analysis showed that miR160 is abundant in developing ovaries. In line with this, its down-regulation perturbed ovary patterning as indicated by the excessive elongation of the proximal ends of mutant ovaries and thinning of the placenta. Following fertilization, these morphological changes led to formation of elongated, pear-shaped fruits reminiscent of those of the tomato ovate mutant. In addition, STTM160-expressing plants displayed abnormal floral organ abscission, and produced leaves, sepals and petals with diminished blades, indicating a requirement for sly-miR160 for these auxin-mediated processes. We found that sly-miR160 depletion was always associated with the up-regulation of SlARF10A, SlARF10B and SlARF17, of which the expression of SlARF10A increased the most. Despite the sly-miR160 legitimate site of SlARF16A, its mRNA levels did not change in response to sly-miR160 down-regulation, suggesting that it may be regulated by a mechanism other than mRNA cleavage. SlARF10A and SlARF17 were previously suggested to function as inhibiting ARFs. We propose that by adjusting the expression of a group of ARF repressors, of which SlARF10A is a primary target, sly-miR160 regulates auxin-mediated ovary patterning as well as floral organ abscission and lateral organ lamina outgrowth.

Control of Anther Cell Differentiation by the Small Protein Ligand TPD1 and Its Receptor EMS1 in Arabidopsis.

A fundamental feature of sexual reproduction in plants and animals is the specification of reproductive cells that conduct meiosis to form gametes, and the associated somatic cells that provide nutrition and developmental cues to ensure successful gamete production. The anther, which is the male reproductive organ in seed plants, produces reproductive microsporocytes (pollen mother cells) and surrounding somatic cells. The microsporocytes yield pollen via meiosis, and the somatic cells, particularly the tapetum, are required for the normal development of pollen. It is not known how the reproductive cells affect the differentiation of these somatic cells, and vice versa. Here, we use molecular genetics, cell biological, and biochemical approaches to demonstrate that TPD1 (TAPETUM DETERMINANT1) is a small secreted cysteine-rich protein ligand that interacts with the LRR (Leucine-Rich Repeat) domain of the EMS1 (EXCESS MICROSPOROCYTES1) receptor kinase at two sites. Analyses of the expressions and localizations of TPD1 and EMS1, ectopic expression of TPD1, experimental missorting of TPD1, and ablation of microsporocytes yielded results suggesting that the precursors of microsporocyte/microsporocyte-derived TPD1 and pre-tapetal-cell-localized EMS1 initially promote the periclinal division of secondary parietal cells and then determine one of the two daughter cells as a functional tapetal cell. Our results also indicate that tapetal cells suppress microsporocyte proliferation. Collectively, our findings show that tapetal cell differentiation requires reproductive-cell-secreted TPD1, illuminating a novel mechanism whereby signals from reproductive cells determine somatic cell fate in plant sexual reproduction.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

The Leu-rich repeat receptor-like kinases SERK1 and SERK2 affect anther cell differentiation as a partners of EMS1 in Arabidopsis. Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

Creating Completely Both Male and Female Sterile Plants by Specifically Ablating Microspore and Megaspore Mother Cells

Although genetically modified (GM) plants have improved commercially important traits, such as biomass and biofuel production, digestibility, bioremediation, ornamental value, and tolerance to biotic and abiotic stresses, there remain economic, political, or social concerns over potential ecological effects of transgene flow from GM plants. The current solution for preventing transgene flow from GM plants is genetically engineering sterility; however, approaches to generating both male and female sterility are limited. In addition, existing strategies for creating sterility lead to loss or modifications of entire flowers or floral organs. Here, we demonstrate that instead of the 1.5-kb promoter, the entire SOLO DANCERS (SDS) gene is required for its meiocyte-specific expression. We then developed an efficient method to specifically ablate microspore and megaspore mother cells using the SDS and BARNASE fusion gene, which resulted in complete sterility in both male and female reproductive organs in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), but did not affect plant growth or development, including the formation of all flower organs. Therefore, our research provides a general and effective tool to prevent transgene flow in GM plants.