NO APICAL MERISTEM (MtNAM) regulates floral organ identity and lateral organ separation in Medicago truncatula

? The CUP-SHAPED COTYLEDON (CUC)/NO APICAL MERISTEM (NAM) family of genes control boundary formation and lateral organ separation, which is critical for proper leaf and flower patterning. However, most downstream targets of CUC/NAM genes remain unclear. ? In a forward screen of the tobacco retrotransposon1 (Tnt1) insertion population in Medicago truncatula, we isolated a weak allele of the no-apical-meristem mutant mtnam-2. Meanwhile, we regenerated a mature plant from the null allele mtnam-1. These materials allowed us to extensively characterize the function of MtNAM and its downstream genes. ? MtNAM is highly expressed in vegetative shoot buds and inflorescence apices, specifically at boundaries between the shoot apical meristem and leaf/flower primordia. Mature plants of the regenerated null allele and the weak allele display remarkable floral phenotypes: floral whorls and organ numbers are reduced and the floral organ identity is compromised. Microarray and quantitative RT-PCR analyses revealed that all classes of floral homeotic genes are down-regulated in mtnam mutants. Mutations in MtNAM also lead to fused cotyledons and leaflets of the compound leaf as well as a defective shoot apical meristem. ? Our results revealed that MtNAM shares the role of CUC/NAM family genes in lateral organ separation and compound leaf development, and is also required for floral organ identity and development.     

SHOOT MERISTEMLESS trafficking controls axillary meristem formation,meristem size and organ boundaries in Arabidopsis

The shoot stem cell niche, contained within the shoot apical meristem (SAM) is maintained in Arabidopsis by the homeodomain protein SHOOT MERISTEMLESS (STM). STM is a mobile protein that traffics cell‐to‐cell, presumably through plasmodesmata. In maize, the STM homolog KNOTTED1 shows clear differences between mRNA and protein localization domains in the SAM. However, the STM mRNA and protein localization domains are not obviously different in Arabidopsis, and the functional relevance of STM mobility is unknown. Using a non‐mobile version of STM (2xNLS‐YFP‐STM), we show that STM mobility is required to suppress axillary meristem formation during embryogenesis, to maintain meristem size, and to precisely specify organ boundaries throughout development. STM and organ boundary genes CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 regulate each other during embryogenesis to establish the embryonic SAM and to specify cotyledon boundaries, and STM controls CUC expression post‐embryonically at organ boundary domains. We show that organ boundary specification by correct spatial expression of CUC genes requires STM mobility in the meristem. Our data suggest that STM mobility is critical for its normal function in shoot stem cell control.     

Polerovirus protein P0 prevents the assembly of small RNA‐containing RISC complexes and leads to degradation of ARGONAUTE1

RNA silencing plays an important role in plants in defence against viruses. To overcome this defence, plant viruses encode suppressors of RNA silencing. The most common mode of silencing suppression is sequestration of double‐stranded RNAs involved in the antiviral silencing pathways. Viral suppressors can also overcome silencing responses through protein–protein interaction. The poleroviral P0 silencing suppressor protein targets ARGONAUTE (AGO) proteins for degradation. AGO proteins are the core component of the RNA‐induced silencing complex (RISC). We found that P0 does not interfere with the slicer activity of pre‐programmed siRNA/miRNA containing AGO1, but prevents de novo formation of siRNA/miRNA containing AGO1. We show that the AGO1 protein is part of a high‐molecular‐weight complex, suggesting the existence of a multi‐protein RISC in plants. We propose that P0 prevents RISC assembly by interacting with one of its protein components, thus inhibiting formation of siRNA/miRNA–RISC, and ultimately leading to AGO1 degradation. Our findings also suggest that siRNAs enhance the stability of co‐expressed AGO1 in both the presence and absence of P0.     

APETALA1 启动子驱动AtIPT4 在转基因拟南芥中表达导致花和花器官发育异常

细胞分裂素对拟南芥(Arab idopsis thal iana)花分生组织细胞的分裂和分化具有重要作用。本研究利用APETALA1(AP1)特异启动子在花分生组织和第1、2轮花器官中表达细胞分裂素合成酶(isopentyl trans ferase, IPT)基因IPT4, 研究细胞分裂素对花和花器官发育的影响。在pAP1::IPT4转基因植株中出现了花密集和花器官数目增多等现象。原位杂交和GUS组织染色结果发现, 在pAP1::IPT4转基因植株中, 花分生组织特征决定基因LEAFY (LFY)与花器官特征决定基因AP1、PISTILLATA (PI )和AGAMOUS (AG)的表达量均有不同程度的提高。研究结果表明在拟南芥中表达pAP1::IPT4影响其花和花器官的正常发育。     

LEAFY, TERMINAL FLOWER1 and AGAMOUS are functionally conserved but do not regulate terminal flowering and floral determinacy in Impatiens balsamina

In Impatiens balsamina a lack of commitment of the meristem during floral development leads to the continuous requirement for a leaf-derived floral signal. In the absence of this signal the meristem reverts to leaf production. Current models for Arabidopsis state that LEAFY (LFY) is central to the integration of floral signals and regulates flowering partly via interactions with TERMINAL FLOWER1 (TFL1) and AGAMOUS (AG). Here we describe Impatiens homologues of LFY, TFL1 and AG (IbLFY, IbTFL1 and IbAG) that are highly conserved at a sequence level and demonstrate homologous functions when expressed ectopically in transgenic Arabidopsis. We relate the expression patterns of IbTFL1 and IbAG to the control of terminal flowering and floral determinacy in Impatiens. IbTFL1 is involved in controlling the phase of the axillary meristems and is expressed in axillary shoots and axillary meristems which produce inflorescences, but not in axillary flowers. It is not involved in maintaining the terminal meristem in either an inflorescence or indeterminate state. Terminal flowering in Impatiens appears therefore to be controlled by a pathway that uses a different integration system than that regulating the development of axillary flowers and branches. The pattern of ovule production in Impatiens requires the meristem to be maintained after the production of carpels. Consistent with this morphological feature IbAG appears to specify stamen and carpel identity, but is not sufficient to specify meristem determinacy in Impatiens.     

Determination of Arabidopsis floral meristem identity by AGAMOUS.

Determinate growth of floral meristems in Arabidopsis requires the function of the floral regulatory gene AGAMOUS (AG). Expression of AG mRNA in the central region of floral meristems relies on the partially overlapping functions of the LEAFY (LFY) and APETALA1 (AP1) genes, which promote initial floral meristem identity. Here, we provide evidence that AG function is required for the final definition of floral meristem identity and that constitutive AG function can promote, independent of LFY and AP1 functions, the determinate floral state in the center of reproductive meristems. Loss-of-function analysis showed that the indeterminate central region of the ag mutant floral meristem undergoes conversion to an inflorescence meristem when long-day-dependent flowering stimulus is removed. Furthermore, gain-of-function analysis demonstrated that ectopic AG function results in precocious flowering and the formation of terminal flowers at apices of both the primary inflorescence and axillary branches of transgenic Arabidopsis plants in which AG expression is under the control of the 35S promoter from cauliflower mosaic virus. Similar phenotypes were also observed in lfy ap1 double mutants carrying a 35S-AG transgene. Together, these results indicate that AG is a principal developmental switch that controls the transition of meristem activity from indeterminate to determinate.     

APETALA1启动子驱动AtIPT4在转基因拟南芥中表达导致花和花器官发育异常

细胞分裂素对拟南芥（Arabidopsis thaliana）花分生组织细胞的分裂和分化具有重要作用。本研究利用APETALA1（AP1）特异启动子在花分生组织和第1、2轮花器官中表达细胞分裂素合成酶（isopentyl transferase,IPT）基因IPT4,研究细胞分裂素对花和花器官发育的影响。在pAP1∷IPT4转基因植株中出现了花密集和花器官数目增多等现象。原位杂交和GUS组织染色结果发现,在pAP1∷IPT4转基因植株中,花分生组织特征决定基因LEAFY（LFY）与花器官特征决定基因AP1、PISTILLATA（PI）和AGAMOUS（AG）的表达量均有不同程度的提高。研究结果表明在拟南芥中表达pAP1∷IPT4影响其花和花器官的正常发育。     

Characterization of an Arabidopsis gene that mediates cytokinin signaling in shoot apical meristem development

Cytokinins are adenine derivatives that regulate numerous plant growth and developmental processes, including apical and floral meristem development, stem growth, leaf senescence, apical dominance, and stress tolerance. However, not much is known about how cytokinin biosynthesis and metabolism is regulated. We identified a novel Arabidopsis gene, ALL, encoding an aldolase-like enzyme that regulates cytokinin signaling. An Arabidopsis mutant, all-1D, in which ALL is activated by the nearby insertion of the 35S enhancer, exhibited extreme dwarfism with rolled, dark-green leaves and reduced apical dominance, symptomatic of cytokinin-overproducing mutants. Consistent with this, ARR4 and ARR5, two representative primary cytokinin-responsive genes, were significantly induced in all-1D. Whereas SHOOT MERISTEMLESS (STM) and KNAT1, which regulate meristem development, were also greatly induced, expression of REV and PHV that regulate lateral organ polarity was inhibited. ALL encodes an aldolase-like enzyme that belongs to the HpcH/HpaI aldolase family in prokaryotes and is down-regulated by exogenous cytokinin, possibly through a negative feedback pathway. We propose that ALL is involved in cytokinin biosynthesis or metabolism and acts as a positive regulator of cytokinin signaling during shoot apical meristem development and determination of lateral organ polarity.     

Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes

The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented.     

A putative lipase gene EXTRA GLUME1 regulates both empty-glume fate and spikelet development in rice

Recent studies have shown that molecular control of inner floral organ identity appears to be largely conserved between monocots and dicots, but little is known regarding the molecular mechanism underlying development of the monocot outer floral organ, a unique floral structure in grasses. In this study, we report the cloning of the rice EXTRA GLUME1 ( EG1 ) gene, a putative lipase gene that specifies empty-glume fate and floral meristem determinacy. In addition to affecting the identity and number of empty glumes, mutations in EG1 caused ectopic floral organs to be formed at each organ whorl or in extra ectopic whorls. Iterative glume-like structures or new floral organ primordia were formed in the presumptive region of the carpel, resulting in an indeterminate floral meristem. EG1 is expressed strongly in inflorescence primordia and weakly in developing floral primordia. We also found that the floral meristem and organ identity gene OsLHS1 showed altered expression with respect to both pattern and levels in the eg1 mutant, and is probably responsible for the pleiotropic floral defects in eg1 . As a putative class III lipase that functionally differs from any known plant lipase, EG1 reveals a novel pathway that regulates rice empty-glume fate and spikelet development.     

CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation

CUP-SHAPED COTYLEDON (CUC)1 encodes members of the NAC family. These are functionally redundant genes that are involved in shoot apical meristem (SAM) formation and cotyledon separation during embryogenesis in Arabidopsis. We analyzed transgenic plants overexpressing CUC1 (35S::CUC1). The cotyledons of these transgenic seedlings regularly had two basal lobes, small and round epidermal cells between the sinuses, and adventitious SAMs on the adaxial surface of this region. This suggests that CUC1 promotes adventitious SAM formation by maintaining epidermal cells in an undifferentiated state. In 35S::CUC1 cotyledons, the class I knotted-like homeobox (KNOX) genes, including SHOOT MERISTEMLESS (STM) and BREVIPEDICELLUS (BP), which are involved in SAM formation and/or maintenance, were ectopically expressed before adventitious SAM formation. In stm mutants, ectopic expression of CUC1 could not induce adventitious SAMs, whereas they continued to be observed in bp mutants. These results suggest that STM, but not BP, is necessary for the formation of adventitious SAMs in 35S::CUC1 cotyledons. Furthermore, we examined the relationship between CUC1 and ASYMMETRIC LEAVES (AS)1 and AS2. The as1 and as2 mutations genetically enhance 35S::CUC1 phenotypes even in the absence of STM function. Interestingly, the as1 mutation can partially rescue the mutant vegetative development phenotypes in the cuc1 cuc2 double mutant. Our results suggest that CUC1 positively regulates SAM formation not only through STM but also through an STM-independent pathway that is negatively regulated by AS1 and AS2.     

Specification of reproductive meristems requires the combined function of SHOOT MERISTEMLESS and floral integrators FLOWERING LOCUS T and FD during Arabidopsis inflorescence development

In Arabidopsis floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene LEAFY. The floral specification activity of FT is dependent upon two related BELL1-like homeobox (BLH) genes PENNYWISE (PNY) and POUND-FOOLISH (PNF) which are required for floral evocation. PNY and PNF interact with a subset of KNOTTED1-LIKE homeobox proteins including SHOOT MERISTEMLESS (STM). Genetic analyses show that these BLH proteins function with STM to specify flowers and internodes during inflorescence development. In this study, experimental evidence demonstrates that the specification of flower and coflorescence meristems requires the combined activities of FT-FD and STM. FT and FD also regulate meristem maintenance during inflorescence development. In plants with reduced STM function, ectopic FT and FD promote the formation of axillary meristems during inflorescence development. Lastly, gene expression studies indicate that STM functions with FT-FD and AGAMOUS-LIKE 24 (AGL24)-SUPPRESSOR OF OVEREXPRESSION OF CONTANS1 (SOC1) complexes to up-regulate flower meristem identity genes during inflorescence development.     

Control of Reproductive Floral Organ Identity Specification in Arabidopsis by the C Function Regulator AGAMOUS

The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.     

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG'mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.