Flowering and determinacy in Arabidopsis

Meristems provide new cells to produce organs throughout the life of a plant, and their continuous activity depends on regulatory genes that balance the proliferation of meristem cells with their recruitment to organogenesis. During flower development, this balance is shifted towards organogenesis, causing the meristem to terminate after producing a genetically determined number of organs. In Arabidopsis, WUSCHEL (WUS) specifies the self-renewing cells at the core of the shoot meristems and is a key target in the control of meristem stability. The development of a determinate floral meristem is initiated by APETALA1/CAULIFLOWER (AP1/CAL) and LEAFY (LFY). The latter activates AGAMOUS (AG), partly in co-operation with WUS. AG then directs the development of the innermost floral organs and at the same time antagonizes WUS to terminate the meristem, although the mechanism of WUS repression remains unknown. All these genes participate in a series of regulatory feedback loops that maintain stable expression patterns or promote sharp developmental transitions. Although the regulators of meristem maintenance and determinacy in Arabidopsis are widely conserved, their interactions may vary in other species.     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Flower development schedule and AGAMOUS‐like gene expression patterns in two morphs of Nigella damascena (Ranunculaceae) differing in floral architecture

Love‐in‐a‐mist (Nigella damascena) is an annual species of Ranunculaceae native to the Mediterranean Basin, characterized by delicate flowers lying on long lacy bracts. Two floral morphs of N. damascena, designated [P] and [T], differ in the identity and number of perianth organs and in the position of the perianth–androecium boundary on the meristem. They both occur in the wild. Here we describe a precise comparative schedule of floral development in the two morphs. We divided the sequence of developmental events affecting the floral meristem into six stages and related them to the height of the elongating stem and to the time elapsed after the beginning of stem elongation. In addition, we characterized the expression pattern of C‐class genes in floral organs of both morphs in an attempt to better characterize the differences between the two floral groundplans. In the [T] morph an expansion of the expression domain of AGAMOUS (AG) paralogues outside the fertile organs was observed, correlating with the change in identity of the inner perianth organs. Expression of AG‐like genes in the sepal‐like organs suggests these are not identical to true sepals at the molecular level. The morpho‐temporal framework we have defined will allow us to compare various gene expression profiles at targeted developmental stages in both morphs, providing further insight into the molecular control of the floral dimorphism in N. damascena and into the processes underlying the transition from a differentiated (bipartite) to an undifferentiated (unipartite) perianth. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 608–619.     

拟南芥WUSCHEL基因在转基因烟草中的超表达(英文)

The Arabidopsis WUSCIHIEL (WUS） gene plays a key role in the specification of the stem cellsin the shoot apical meristem (SAM). A cDNA of WUShas been amplified with the RT-PCR approach fromArabidopsis. The plant overexpression vector was constructed. It was driven by a dual enhanced CaMV35Spromoter. The construct was transformed into tobacco (Nicotiana tabacum L.) via Agrobacterium mediation.Dramatic phenotypic changes appeared in the WUS overexpression transgenic plants. Aberrant celldivisions and ectopic organogenesis could be found in almost every aerial parts of the transgenic tobaccoexcept the meristems and the inner two floral whorls. The data showed a highly conserved function of WUSin tobacco, and suggested that WUS is involved in organogenesis. The leaves were malformed, whichstrongly matched those only described previously for plants grown in the presence of polar auxin transportinhibitors. It suggested a possible function of WUS in leaf development. These results provide usefulinformation for functional analysis of WUS and important biotechnological implication as well.     

拟南芥WUSCHEL基因在转基因烟草中的超表达

WUSCHEL(WUS)是近年报道的一个重要的干细胞调控基因.本实验用RT-PCR技术从拟南芥(Arabidopsisthaliana L.)中克隆到其cDNA并构建了双增强的CaMV3 5S启动子驱动的超表达载体pBKB.借助农杆菌(Agrobacterium tumefaciens)介导转化烟草(Nicotiana tabacum L.),获得转基因植株.PCR和RT-PCR鉴定分别证明,外源WUS已整合到烟草基因组并已表达.转基因烟草地上部分出现大量异位增生的突起,扫描电镜观察表明:突起部分的细胞与分生组织细胞相似,部分突起能够发育为叶芽、花芽,表明WUS超表达引起烟草细胞异常分裂并在已分化组织中重新启动了器官形成.茎尖和花的内两轮器官没有上述变化.结合拟南芥的有关研究,推测烟草中可能也存在类似拟南芥WUS和其阻抑蛋白CLAVATA3、AGAMOUS间的反馈调节机制.转基因烟草叶发育表型变化明显,与生长素极性运输受抑制引起的表型相似,因此,作为生长点调控基因,WUS可能通过生长素对叶的发育进行调控.本研究为WUS基因的功能分析和有关生物技术应用提供了有意义的信息.     

The rice FISH BONE gene encodes a tryptophan aminotransferase,which affects pleiotropic auxin‐related processes

Auxin is a fundamental plant hormone and its localization within organs plays pivotal roles in plant growth and development. Analysis of many Arabidopsis mutants that were defective in auxin biosynthesis revealed that the indole‐3‐pyruvic acid (IPA) pathway, catalyzed by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) families, is the major biosynthetic pathway of indole‐3‐acetic acid (IAA). In contrast, little information is known about the molecular mechanisms of auxin biosynthesis in rice. In this study, we identified a auxin‐related rice mutant, fish bone (fib). FIB encodes an orthologue of TAA genes and loss of FIB function resulted in pleiotropic abnormal phenotypes, such as small leaves with large lamina joint angles, abnormal vascular development, small panicles, abnormal organ identity and defects in root development, together with a reduction in internal IAA levels. Moreover, we found that auxin sensitivity and polar transport activity were altered in the fib mutant. From these results, we suggest that FIB plays a pivotal role in IAA biosynthesis in rice and that auxin biosynthesis, transport and sensitivity are closely interrelated.     

Yucasin is a potent inhibitor of YUCCA,a key enzyme in auxin biosynthesis

Indole‐3–acetic acid (IAA), an auxin plant hormone, is biosynthesized from tryptophan. The indole‐3–pyruvic acid (IPyA) pathway, involving the tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA biosynthetic pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA, and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5–(4–chlorophenyl)‐4H‐1,2,4–triazole‐3–thiol (yucasin) as a potent inhibitor of IAA biosynthesis in YUC‐expressing coleoptile tips. Enzymatic analysis of recombinant AtYUC1‐His suggested that yucasin strongly inhibited YUC1‐His activity against the substrate IPyA in a competitive manner. Phenotypic analysis of Arabidopsis YUC1 over‐expression lines (35S::YUC1) demonstrated that yucasin acts in IAA biosynthesis catalyzed by YUC. In addition, 35S::YUC1 seedlings showed resistance to yucasin in terms of root growth. A loss‐of‐function mutant of TAA1, sav3–2, was hypersensitive to yucasin in terms of root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor l –kynurenine acted additively in Arabidopsis seedlings, producing a phenotype similar to yucasin‐treated sav3–2 seedlings, indicating the importance of IAA biosynthesis via the IPyA pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUC enzymes that offers an effective tool for analyzing the contribution of IAA biosynthesis via the IPyA pathway to plant development and physiological processes.     

Termination of stem cell maintenance in Arabidopsis floral meristems by interactions between WUSCHEL and AGAMOUS.

Floral meristems and shoot apical meristems (SAMs) are homologous, self-maintaining stem cell systems. Unlike SAMs, floral meristems are determinate, and stem cell maintenance is abolished once all floral organs are initiated. To investigate the underlying regulatory mechanisms, we analyzed the interactions between WUSCHEL (WUS), which specifies stem cell identity, and AGAMOUS (AG), which is required for floral determinacy. Our results show that repression of WUS by AG is essential for terminating the floral meristem and that WUS can induce AG expression in developing flowers. Together, this suggests that floral determinacy depends on a negative autoregulatory mechanism involving WUS and AG, which terminates stem cell maintenance.     

Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid‐mediated defenses

Auxin is a key plant growth regulator that also impacts plant–pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole‐3‐acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector‐triggered immunity was active in YUC1‐overexpressing plants, and we observed only minor effects on SA levels and SA‐mediated responses. Furthermore, a plant line carrying both the YUC1‐overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA‐mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA‐mediated defenses.     

Patterns and Polarity in Floral Meristem and Floral Organ Initiation

The initial event in plant floral organogenesis is bract specification, followed by floral meristem (FM) initiation in bract axils, but initiation signals and the interplay between both lateral organs remain unelucidated. Floral organs are initiated on the flanks of the outgrowing FM and the enormous diversity in floral morphology throughout the plant kingdom reflects variations in organ position, meristy and ontogeny. Classical models of floral development have focused on Arabidopsis, which has mostly actinomorphic flowers, and Antirrhinum, which exhibits zygomorphy, although neither species is typical or representative of angiosperm flower diversity. Although the ABCE model defines a centripetal model of organ identity establishment in different whorls, the characterization of floral organ initiation in many species has relied on their morphological appearance, due to a lack of founder cell-specific markers. Recent progress in early Arabidopsis floral development using histology, molecular markers and mutants has led to refinements of existing floral organ initiation paradigms. In Arabidopsis, sepals initiate unidirectionally, in a temporal window characterized by the absence of CLAVATA3 and WUSCHEL stem cell markers and are partly dependent on PRESSED FLOWER function, whereas initiation of inner-whorl organs occurs centripetally. Arabidopsis mutants reveal that the FM is highly polarized along an ab-/adaxial axis and a comparison of floral development in Arabidopsis and Antirrhinum suggests that heterochrony of conserved gene functions has been evolutionarily adaptive.

This review discusses current views on FM and organ specification signals, the gene regulatory networks that underlie floral meristem polarity, and analogies between the development of floral and leaf primordia as lateral organs. Alternative stem-cell proliferation mechanisms and the bifurcation of founder cell populations can help to explain the diversity in floral diversity throughout the plant kingdom and underpin comparative evolutionary biology and macroevolution. An analysis of plants with divergent body plans at the level of organ specification is urgently needed.     

植物干细胞决定基因WUS的研究进展

WUS(WUSCHEL)基因编码一转录因子,它的存在使周围细胞具有干细胞的特征,与之相关的信号系统近年逐步被阐明.在茎尖分生组织内WUS和CLV(CLAVATA)之间形成一个反馈调节环,使得干细胞保持自我更新,维持茎尖的顶端优势.在胚胎分生组织内,CLV3的表达只依赖于WUS的存在,然而在胚以后的发育中,CLV3的表达受到WUS和STM(SHOOTMERISTEMLESS)的双重调节,启动器官发生.在花分生组织中,WUS和LFY(LEAFY)共同激活AG(AGAMOUS)基因的表达,WUS受AG的反馈抑制.由WUS建立的信号体系还参与胚珠的发育.当WUS蛋白和生长素共存时,可以高效启动体细胞胚的发生.细胞对WUS信号的感应性与细胞所处的微环境有关,WUS在不同环境条件下可以启动不同的下游基因表达.     

Benzylaminopurine induces phenocopies of floral meristem and organ identity mutants in wild-typeArabidopsis plants

Arabidopsis thaliana (L.) Heynh. has been used as a model system to investigate the regulatory genes that control and coordinate the determination, differentiation and morphogenesis of the floral meristem and floral organs. We show here that benzylaminopurine (BAP), a cytokinin, influences flower development inArabidopsis and induces partial phenocopies of known floral homeotic mutants. Application of BAP to wild-type inflorescences at three developmental stages results in: (i) increase in floral organ number; (ii) formation of abnormal floral organs and (iii) induction of secondary floral buds in the axils of sepals. These abnormalities resemble the phenotypes of mutants,clv1 (increase in organ number),ap1,ap2,ap3 (abnormal floral organs) andap1 (secondary floral buds in the axils of first-whorl organs). In addition, BAP induces secondary floral buds in the axils of perianth members ofapt2-6, ap3-1 andag mutants, and accentuates the phenotype of theapt2-1 mutant to resemble theapt2-6 mutant. These observations suggest that exogenous BAP suppresses the normal functioning of the genes for floral meristem identity and thereby affects flower development and the later stages of floral organ differentiation.Abbreviations BAP N6-benzylaminopurine - CK cytokinin     

Allelic Analyses of the Arabidopsis YUC1 Locus Reveal Residues and Domains Essential for the Functions of YUC Family of Flavin Monooxygenases

Flavin monooxygenases(FMOs) play critical roles in plant growth and development by synthesizing auxin and other signaling molecules.However,the structure and function relationship within plant FMOs is not understood.Here we defined the important residues and domains of the Arabidopsis YUC1 FMO,a key enzyme in auxin biosynthesis.We previously showed that simultaneous inactivation of YUC1 and its homologue YUC4 caused severe defects in vascular and floral development.We mutagenized the yuc4 mutant and screene...     

Cloning and Biochemical Characterization of <Emphasis Type="Italic">ToFZY</Emphasis>, a Tomato Gene Encoding a Flavin Monooxygenase Involved in a Tryptophan-dependent Auxin Biosynthesis Pathway

Indole-3-acetic acid (IAA), the main endogenous auxin, has been known for decades to be a key regulator for plant growth and development. Multiple routes have been proposed for IAA biosynthesis but physiologic roles or relevance of the different routes are still unclear. Recently, four members of the Arabidopsis thaliana YUC gene family have been implicated in an additional requirement of IAA involved in floral organ and vascular tissue formation. The loss-of-function yuc1yuc4 double mutants in Arabidopsis displayed phenotypes similar to the previously described loss-of-function floozy mutants in petunia (fzy). Moreover, it has been demonstrated that YUC1 encodes a flavin monooxygenase (FMO) that catalyzes a rate-limiting step of a tryptophan-dependent auxin biosynthesis pathway: the conversion of tryptamine to N-hydroxyl-tryptamine. Here we report on the genetic study of ToFZY, the putative tomato ortholog of YUC4 and FZY, including gene and cDNA sequence comparison and a preliminary expression analysis. In addition, we describe a novel conserved amino acid motif that may be considered a hallmark potentially useful for the identification of new YUC-like FMOs. We also demonstrate that ToFZY encodes a protein with the same enzymatic activity as YUC1. Finally, we provide evidence suggesting that the ToFZY gene belongs to a multigenic family whose members may exhibit a temporal and spatial specialization similar to that described in A. thaliana.     

An APETALA3 homolog controls both petal identity and floral meristem patterning in Nigella damascena L. (Ranunculaceae)

Flower architecture mutants provide a unique opportunity to address the genetic origin of flower diversity. Here we study a naturally occurring floral dimorphism in Nigella damascena (Ranunculaceae), involving replacement of the petals by numerous sepal‐like and chimeric sepal/stamen organs. We performed a comparative study of floral morphology and floral development, and characterized the expression of APETALA3 and PISTILLATA homologs in both morphs. Segregation analyses and gene silencing were used to determine the involvement of an APETALA3 paralog (NdAP3–3) in the floral dimorphism. We demonstrate that the complex floral dimorphism is controlled by a single locus, which perfectly co‐segregates with the NdAP3–3 gene. This gene is not expressed in the apetalous morph and exhibits a particular expression dynamic during early floral development in the petalous morph. NdAP3–3 silencing in petalous plants perfectly phenocopies the apetalous morph. Our results show that NdAP3–3 is fully responsible for the complex N. damascena floral dimorphism, suggesting that it plays a role not only in petal identity but also in meristem patterning, possibly through regulation of perianth organ number and the perianth/stamen boundary.