Growth dynamics of the Arabidopsis fruit is mediated by cell expansion.

Fruit have evolved a sophisticated tissue and cellular architecture to secure plant reproductive success. Postfertilization growth is perhaps the most dramatic event during fruit morphogenesis. Several studies have proposed that fertilized ovules and developing seeds initiate signaling cascades to coordinate and promote the growth of the accompanying fruit tissues. This dynamic process allows the fruit to conspicuously increase its size and acquire its final shape and means for seed dispersal. All these features are key for plant survival and crop yield. Despite its importance, we lack a high-resolution spatiotemporal map of how postfertilization fruit growth proceeds at the cellular level. In this study, we have combined live imaging, mutant backgrounds in which fertilization can be controlled, and computational modeling to monitor and predict postfertilization fruit growth in Arabidopsis We have uncovered that, unlike leaves, sepals, or roots, fruit do not exhibit a spatial separation of cell division and expansion domains; instead, there is a separation into temporal stages with fertilization as the trigger for transitioning to cell expansion, which drives postfertilization fruit growth. We quantified the coordination between fertilization and fruit growth by imaging no transmitting tract (ntt) mutants, in which fertilization fails in the bottom half of the fruit. By combining our experimental data with computational modeling, we delineated the mobility properties of the seed-derived signaling cascades promoting growth in the fruit. Our study provides the basis for generating a comprehensive understanding of the molecular and cellular mechanisms governing fruit growth and shape.

Gibberellins negatively modulate ovule number in plants.

Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.

Ovule identity mediated by pre-mRNA processing in Arabidopsis.

Ovules are fundamental for plant reproduction and crop yield as they are the precursors of seeds. Therefore, ovule specification is a critical developmental program. In Arabidopsis thaliana, ovule identity is redundantly conferred by the homeotic D-class genes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), phylogenetically related to the MADS-domain regulatory gene AGAMOUS (AG), essential in floral organ specification. Previous studies have shown that the HUA-PEP activity, comprised of a suite of RNA-binding protein (RBP) encoding genes, regulates AG pre-mRNA processing and thus flower patterning and organ identity. Here, we report that the HUA-PEP activity additionally governs ovule morphogenesis. Accordingly, in severe hua-pep backgrounds ovules transform into flower organ-like structures. These homeotic transformations are most likely due to the dramatic reduction in SHP1, SHP2 and STK activity. Our molecular and genome-wide profiling strategies revealed the accumulation of prematurely terminated transcripts of D-class genes in hua-pep mutants and reduced amounts of their respective functional messengers, which points to pre-mRNA processing misregulation as the origin of the ovule developmental defects in such backgrounds. RNA processing and transcription are coordinated by the RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD). Our results show that HUA-PEP activity members can interact with the CTD regulator C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 (CPL1), supporting a co-transcriptional mode of action for the HUA-PEP activity. Our findings expand the portfolio of reproductive developmental programs in which HUA-PEP activity participates, and further substantiates the importance of RNA regulatory mechanisms (pre-mRNA co-transcriptional regulation) for correct gene expression during plant morphogenesis.

The bHLH transcription factor SPATULA enables cytokinin signaling, and both activate auxin biosynthesis and transport genes at the medial domain of the gynoecium.

Fruits and seeds are the major food source on earth. Both derive from the gynoecium and, therefore, it is crucial to understand the mechanisms that guide the development of this organ of angiosperm species. In Arabidopsis, the gynoecium is composed of two congenitally fused carpels, where two domains: medial and lateral, can be distinguished. The medial domain includes the carpel margin meristem (CMM) that is key for the production of the internal tissues involved in fertilization, such as septum, ovules, and transmitting tract. Interestingly, the medial domain shows a high cytokinin signaling output, in contrast to the lateral domain, where it is hardly detected. While it is known that cytokinin provides meristematic properties, understanding on the mechanisms that underlie the cytokinin signaling pattern in the young gynoecium is lacking. Moreover, in other tissues, the cytokinin pathway is often connected to the auxin pathway, but we also lack knowledge about these connections in the young gynoecium. Our results reveal that cytokinin signaling, that can provide meristematic properties required for CMM activity and growth, is enabled by the transcription factor SPATULA (SPT) in the medial domain. Meanwhile, cytokinin signaling is confined to the medial domain by the cytokinin response repressor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFERASE 6 (AHP6), and perhaps by ARR16 (a type-A ARR) as well, both present in the lateral domains (presumptive valves) of the developing gynoecia. Moreover, SPT and cytokinin, probably together, promote the expression of the auxin biosynthetic gene TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) and the gene encoding the auxin efflux transporter PIN-FORMED 3 (PIN3), likely creating auxin drainage important for gynoecium growth. This study provides novel insights in the spatiotemporal determination of the cytokinin signaling pattern and its connection to the auxin pathway in the young gynoecium.

A role for the gene regulatory module microRNA172/TARGET OF EARLY ACTIVATION TAGGED 1/FLOWERING LOCUS T (miRNA172/TOE1/FT) in the feeding sites induced by Meloidogyne javanica in Arabidopsis thaliana.

Root knot nematodes (RKNs) penetrate into the root vascular cylinder, triggering morphogenetic changes to induce galls, de novo formed 'pseudo-organs' containing several giant cells (GCs). Distinctive gene repression events observed in early gall/GCs development are thought to be mediated by post-transcriptional silencing via microRNAs (miRNAs), a process that is far from being fully characterized. Arabidopsis thaliana backgrounds with altered activities based on target 35S::MIMICRY172 (MIM172), 35S::TARGET OF EARLY ACTIVATION TAGGED 1 (TOE1)-miR172-resistant (35S::TOE1R ) and mutant (flowering locus T-10 (ft-10)) lines were used for functional analysis of nematode infective and reproductive parameters. The GUS-reporter lines, MIR172A-E::GUS, treated with auxin (IAA) and an auxin-inhibitor (a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA)), together with the MIR172C AuxRE::GUS line with two mutated auxin responsive elements (AuxREs), were assayed for nematode-dependent gene expression. Arabidopsis thaliana backgrounds with altered expression of miRNA172, TOE1 or FT showed lower susceptibility to the RKNs and smaller galls and GCs. MIR172C-D::GUS showed restricted promoter activity in galls/GCs that was regulated by auxins through auxin-responsive factors. IAA induced their activity in galls while PEO-IAA treatment and mutations in AuxRe motifs abolished it. The results showed that the regulatory module miRNA172/TOE1/FT plays an important role in correct GCs and gall development, where miRNA172 is modulated by auxins.

K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis.

Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.

A regulated auxin minimum is required for seed dispersal in Arabidopsis.

Local hormone maxima are essential for the development of multicellular structures and organs. For example, steroid hormones accumulate in specific cell types of the animal fetus to induce sexual differentiation and concentration peaks of the plant hormone auxin direct organ initiation and mediate tissue patterning. Here we provide an example of a regulated local hormone minimum required during organogenesis. Our results demonstrate that formation of a local auxin minimum is necessary for specification of the valve margin separation layer where Arabidopsis fruit opening takes place. Consequently, ectopic production of auxin, specifically in valve margin cells, leads to a complete loss of proper cell fate determination. The valve margin identity factor INDEHISCENT (IND) is responsible for forming the auxin minimum by coordinating auxin efflux in separation-layer cells. We propose that the simplicity of formation and maintenance make local hormone minima particularly well suited to specify a small number of cells such as the stripes at the valve margins.

Antagonistic gene activities determine the formation of pattern elements along the mediolateral axis of the Arabidopsis fruit.

The Arabidopsis fruit mainly consists of a mature ovary that shows three well defined territories that are pattern elements along the mediolateral axis: the replum, located at the medial plane of the flower, and the valve and the valve margin, both of lateral nature. JAG/FIL activity, which includes the combined functions of JAGGED (JAG), FILAMENTOUS FLOWER (FIL), and YABBY3 (YAB3), contributes to the formation of the two lateral pattern elements, whereas the cooperating genes BREVIPEDICELLUS (BP) and REPLUMLESS (RPL) promote replum development. A recent model to explain pattern formation along the mediolateral axis hypothesizes that JAG/FIL activity and BP/RPL function as antagonistic lateral and medial factors, respectively, which tend to repress each other. In this work, we demonstrate the existence of mutual exclusion mechanisms between both kinds of factors, and how this determines the formation and size of the three territories. Medial factors autonomously constrain lateral factors so that they only express outside the replum, and lateral factors negatively regulate the medially expressed BP gene in a non-autonomous fashion to ensure correct replum development. We also have found that ASYMMETRIC LEAVES1 (AS1), previously shown to repress BP both in leaves and ovaries, collaborates with JAG/FIL activity, preventing its repression by BP and showing synergistic interactions with JAG/FIL activity genes. Therefore AS gene function (the function of the interacting genes AS1 and AS2) has been incorporated in the model as a new lateral factor. Our model of antagonistic factors provides explanation for mutant fruit phenotypes in Arabidopsis and also may help to understand natural variation of fruit shape in Brassicaceae and other species, since subtle changes in gene expression may cause conspicuous changes in the size of the different tissue types.

The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit.

The Arabidopsis fruit forms a seedpod that develops from the fertilized gynoecium. It is mainly comprised of an ovary in which three distinct tissues can be differentiated: the valves, the valve margins and the replum. Separation of cells at the valve margin allows for the valves to detach from the replum and thus dispersal of the seeds. Valves and valve margins are located in lateral positions whereas the replum is positioned medially and retains meristematic properties resembling the shoot apical meristem (SAM). Members of the WUSCHEL-related homeobox family have been involved in stem cell maintenance in the SAM, and within this family, we found that WOX13 is expressed mainly in meristematic tissues including the replum. We also show that wox13 loss-of-function mutations reduce replum size and enhance the phenotypes of mutants affected in the replum identity gene RPL. Conversely, misexpression of WOX13 produces, independently from BP and RPL, an oversized replum and valve defects that closely resemble those of mutants in JAG/FIL activity genes. Our results suggest that WOX13 promotes replum development by likely preventing the activity of the JAG/FIL genes in medial tissues. This regulation seems to play a role in establishing the gradient of JAG/FIL activity along the medio-lateral axis of the fruit critical for proper patterning. Our data have allowed us to incorporate the role of WOX13 into the regulatory network that orchestrates fruit patterning.

HALF FILLED promotes reproductive tract development and fertilization efficiency in Arabidopsis thaliana.

Successful fertilization in angiosperms requires the growth of pollen tubes through the female reproductive tract as they seek out unfertilized ovules. In Arabidopsis, the reproductive tract begins with the stigma, where pollen grains initially adhere, and extends through the transmitting tract of the style and ovary. In wild-type plants, cells within the transmitting tract produce a rich extracellular matrix and undergo programmed cell death to facilitate pollen movement. Here, we show that the HAF, BEE1 and BEE3 genes encode closely related bHLH transcription factors that act redundantly to specify reproductive tract tissues. These three genes are expressed in distinct but overlapping patterns within the reproductive tract, and in haf bee1 bee3 triple mutants extracellular matrix formation and cell death fail to occur within the transmitting tract. We used a minimal pollination assay to show that HAF is necessary and sufficient to promote fertilization efficiency. Our studies further show that HAF expression depends on the NTT gene and on an auxin signaling pathway mediated by the ARF6, ARF8 and HEC genes.

A novel role for the floral homeotic gene APETALA2 during Arabidopsis fruit development.

The majority of the Arabidopsis fruit comprises an ovary with three primary tissue types: the valves, the replum and the valve margins. The valves, which are derived from the ovary walls, are separated along their entire length by the replum. The valve margin, which consists of a separation layer and a lignified layer, forms as a narrow stripe of cells at the valve-replum boundaries. The valve margin identity genes are expressed at the valve-replum boundary and are negatively regulated by FUL and RPL in the valves and replum, respectively. In ful rpl double mutants, the valve margin identity genes become ectopically expressed, and, as a result, the entire outer surface of the ovary takes on valve margin identity. We carried out a genetic screen in this sensitized genetic background and identified a suppressor mutation that restored replum development. Surprisingly, we found that the corresponding suppressor gene was AP2, a gene that is well known for its role in floral organ identity, but whose role in Arabidopsis fruit development had not been previously described. We found that AP2 acts to prevent replum overgrowth by negatively regulating BP and RPL, two genes that normally act to promote replum formation. We also determined that AP2 acts to prevent overgrowth of the valve margin by repressing valve margin identity gene expression. We have incorporated AP2 into the current genetic network controlling fruit development in Arabidopsis.

Brassicaceae INDEHISCENT genes specify valve margin cell fate and repress replum formation.

Members of the Brassicaceae family, including Arabidopsis thaliana and oilseed rape (Brassica napus), produce dry fruits that open upon maturity along a specialised tissue called the valve margin. Proper development of the valve margin in Arabidopsis is dependent on the INDEHISCENT (IND) gene, the role of which in genetic and hormonal regulation has been thoroughly characterised. Here we perform phylogenetic comparison of IND genes in Arabidopsis and Brassica to identify conserved regulatory sequences that are responsible for specific expression at the valve margin. In addition we have taken a comparative development approach to demonstrate that the BraA.IND.a and BolC.IND.a genes from B. rapa and B. oleracea share identical function with Arabidopsis IND since ethyl methanesulphonate (EMS) mutant alleles and silenced transgenic lines have valve margin defects. Furthermore we show that the degree of these defects can be fine-tuned for crop improvement. Wild-type Arabidopsis produces an outer replum composed of about six cell files at the medial region of the fruits, whereas Brassica fruits lack this tissue. A strong loss-of-function braA.ind.a mutant gained outer replum tissue in addition to its defect in valve margin development. An enlargement of replum size was also observed in the Arabidopsis ind mutant suggesting a general role of Brassicaceae IND genes in preventing valve margin cells from adopting replum identity.

The formation and function of the female reproductive tract in flowering plants.

In angiosperms, sexual reproduction requires a sperm cell, contained within a pollen tube, to fertilize the egg cell. The pollen tubes are capable of growth but have a difficult journey, as egg cells are buried within the ovary of the carpel. Several tissues, known collectively as the reproductive tract, develop within the carpel to facilitate the journey of the pollen tube. The genes involved in the formation and function of the reproductive tract have largely remained a mystery but are crucial for successful fertilization. This review summarizes recent advances in our understanding of the genetic control of reproductive tract development.

The NTT gene is required for transmitting-tract development in carpels of Arabidopsis thaliana.

BACKGROUND: The majority of pollen-tube growth in Arabidopsis occurs in specialized tissue called the transmitting tract. Little is currently known about how the transmitting tract functions because of a lack of mutants affecting its development. We have identified such a mutant and we used it to investigate aspects of pollen-tube growth. RESULTS: Reverse genetics was used to identify mutations in a gene, No Transmitting Tract (NTT), encoding a C2H2/C2HC zinc finger transcription factor specifically expressed in the transmitting tract. The ntt mutants have a negative effect on transmitting-tract development. Stage-specific analysis of transmitting-tract development was carried out and was correlated with investigations of pollen-tube behavior. In ntt mutants, pollen tubes grow more slowly and/or terminate prematurely, and lateral divergence is accentuated over apical-to-basal movement. Normal transmitting-tract development is shown to involve a process of programmed cell death (PCD) that is facilitated by, but does not depend upon, pollination. CONCLUSIONS: This is the first report of a gene that is specifically required for transmitting-tract development in Arabidopsis. Mutations in NTT cause reduced fertility by severely inhibiting pollen-tube movement. The data support the idea that the function of the transmitting tract is to increase fertilization efficiency, particularly in the lower half of the ovary. This occurs by facilitating pollen-tube growth through differentiation and then death of transmitting-tract cells.

Assessing the redundancy of MADS-box genes during carpel and ovule development.

Carpels are essential for sexual plant reproduction because they house the ovules and subsequently develop into fruits that protect, nourish and ultimately disperse the seeds. The AGAMOUS (AG) gene is necessary for plant sexual reproduction because stamens and carpels are absent from ag mutant flowers. However, the fact that sepals are converted into carpelloid organs in certain mutant backgrounds even in the absence of AG activity indicates that an AG-independent carpel-development pathway exists. AG is a member of a monophyletic clade of MADS-box genes that includes SHATTERPROOF1 (SHP1), SHP2 and SEEDSTICK (STK), indicating that these four genes might share partly redundant activities. Here we show that the SHP genes are responsible for AG-independent carpel development. We also show that the STK gene is required for normal development of the funiculus, an umbilical-cord-like structure that connects the developing seed to the fruit, and for dispersal of the seeds when the fruit matures. We further show that all four members of the AG clade are required for specifying the identity of ovules, the landmark invention during the course of vascular plant evolution that enabled seed plants to become the most successful group of land plants.

Inflorescence Meristem Fate Is Dependent on Seed Development and FRUITFULL in Arabidopsis thaliana.

After a vegetative phase, plants initiate the floral transition in response to both environmental and endogenous cues to optimize reproductive success. During this process, the vegetative shoot apical meristem (SAM), which was producing leaves and branches, becomes an inflorescence SAM and starts producing flowers. Inflorescences can be classified in two main categories, depending on the fate of the inflorescence meristem: determinate or indeterminate. In determinate inflorescences, the SAM differentiates directly, or after the production of a certain number of flowers, into a flower, while in indeterminate inflorescences the SAM remains indeterminate and produces continuously new flowers. Even though indeterminate inflorescences have an undifferentiated SAM, the number of flowers produced by a plant is not indefinite and is characteristic of each species, indicating that it is under genetic control. In Arabidopsis thaliana and other species with indeterminate inflorescences, the end of flower production occurs by a regulated proliferative arrest of inflorescence meristems on all reproductive branches that is reminiscent of a state of induced dormancy and does not involve the determination of the SAM. This process is controlled genetically by the FRUITFULL-APETALA2 (FUL-AP2) pathway and by a correlative control exerted by the seeds through a mechanism not well understood yet. In the absence of seeds, meristem proliferative arrest does not occur, and the SAM remains actively producing flowers until it becomes determinate, differentiating into a terminal floral structure. Here we show that the indeterminate growth habit of Arabidopsis inflorescences is a facultative condition imposed by the meristematic arrest directed by FUL and the correlative signal of seeds. The terminal differentiation of the SAM when seed production is absent correlates with the induction of AGAMOUS expression in the SAM. Moreover, terminal flower formation is strictly dependent on the activity of FUL, as it was never observed in ful mutants, regardless of the fertility of the plant or the presence/absence of the AG repression exerted by APETALA2 related factors.

Genetic control of meristem arrest and life span in Arabidopsis by a FRUITFULL-APETALA2 pathway.

Monocarpic plants have a single reproductive cycle in their lives, where life span is determined by the coordinated arrest of all meristems, or global proliferative arrest (GPA). The molecular bases for GPA and the signaling mechanisms involved are poorly understood, other than systemic cues from developing seeds of unknown nature. Here we uncover a genetic pathway regulating GPA in Arabidopsis that responds to age-dependent factors and acts in parallel to seed-derived signals. We show that FRUITFULL (FUL), a MADS-box gene involved in flowering and fruit development, has a key role in promoting meristem arrest, as GPA is delayed and fruit production is increased in ful mutants. FUL directly and negatively regulates APETALA2 expression in the shoot apical meristem and maintains the temporal expression of WUSCHEL which is an essential factor for meristem maintenance.

Vascular patterning: xylem or phloem?

Xylem and phloem are the major conduits for the transport of water and solutes through the plant. Recent work has helped to elucidate the mechanisms that determine the identity and arrangement of these two tissues.

Floral development: an ABC gene chips in downstream.

In flower development, specification of stamens and carpels requires the AGAMOUS gene. A recent study has now shown that AGAMOUS also plays more specific roles in the regional activation of sporocyte formation.

The role of the REPLUMLESS homeodomain protein in patterning the Arabidopsis fruit.

The outside of the Arabidopsis thaliana fruit consists of three principal tissues: the valves or seedpod walls, the replum or central ridge between the valves, and the valve margins where the valves separate from the replum to disperse the seeds. Previous studies have shown that valve margin formation is specified by the SHATTERPROOF MADS-box transcription factors and that valve development is controlled by the FRUITFULL MADS-box transcription factor. FRUITFULL negatively regulates SHATTERPROOF to prevent the valves from adopting a valve margin cell fate. Here we identify a gene called REPLUMLESS that is required for replum development. REPLUMLESS encodes a homeodomain protein that prevents replum cells from adopting a valve margin cell fate by negatively regulating expression of the SHATTERPROOF genes. Both REPLUMLESS and FRUITFULL are required to limit SHATTERPROOF expression to a narrow stripe of cells so that the valve margin differentiates precisely at the valve/replum boundary.