Comparative morphoanatomy and transcriptomic analyses reveal key factors controlling floral trichome development in Aristolochia (Aristolochiaceae).

Trichomes are specialized epidermal cells in aerial plant parts. Trichome development proceeds in three stages, determination of cell fate, specification, and morphogenesis. Most genes responsible for these processes have been identified in the unicellular branched leaf trichomes from the model Arabidopsis thaliana. Less is known about the molecular basis of multicellular trichome formation across flowering plants, especially those formed in floral organs of early diverging angiosperms. Here, we aim to identify the genetic regulatory network (GRN) underlying multicellular trichome development in the kettle-shaped trap flowers of Aristolochia (Aristolochiaceae). We selected two taxa for comparison, A. fimbriata, with trichomes inside the perianth, which play critical roles in pollination, and A. macrophylla, lacking specialized trichomes in the perianth. A detailed morphoanatomical characterization of floral epidermis is presented for the two species. We compared transcriptomic profiling at two different developmental stages in the different perianth portions (limb, tube, and utricle) of the two species. Moreover, we present a comprehensive expression map for positive regulators and repressors of trichome development, as well as cell cycle regulators. Our data point to extensive modifications in gene composition, expression, and putative roles in all functional categories when compared with model species. We also record novel differentially expressed genes (DEGs) linked to epidermis patterning and trichome development. We thus propose the first hypothetical genetic regulatory network (GRN) underlying floral multicellular trichome development in Aristolochia, and pinpoint key factors responsible for the presence and specialization of floral trichomes in phylogenetically distant species of the genus.

The floral repressors TEMPRANILLO1 and 2 modulate salt tolerance by regulating hormonal components and photo-protection in Arabidopsis.

Members of the plant specific RAV family of transcription factors regulate several developmental and physiological processes. In the model plant Arabidopsis thaliana, the RAV TEMPRANILLO 1 (TEM1) and TEM2 control important phase changes such as the juvenile to adult and the vegetative to reproductive transitions. Besides their known regulatory function in plant development, a transcriptomics analysis of transgenic plants overexpressing TEM1 also revealed overrepresentation of Gene Ontology (GO) categories related to abiotic stress responses. Therefore, to investigate the biological relevance of these TEM-dependent transcriptomic changes and elucidate whether TEMs contribute to the modulation of plant growth in response to salinity, we analyzed the behavior of TEM gain and loss of function mutants subjected to mild and high salt stresses at different development stages. With respect to increasing salinity, TEM overexpressing plants were hypersensitive whereas the tem1 tem2 double mutants were more tolerant. Precisely, tem1 tem2 mutants germinated and flowered faster than the wild-type plants under salt stress conditions. Also, tem1 tem2 plants showed a delay in salt-induced leaf senescence, possibly as a consequence of downregulation of jasmonic acid biosynthesis genes. Besides a shorter life cycle and delayed senescence, tem1 tem2 mutants appeared to be better suited to withstand oxidative stress as they accumulated higher levels of α-tocopherol (an important antioxidant metabolite) and displayed a slower degradation of photosynthetic pigments. Taken together, our studies suggest novel and crucial roles for TEM in adaptive growth as they modulate plant development in response to environmental changes such as increasing soil salinity.

Genes of the RAV Family Control Heading Date and Carpel Development in Rice.

In plants, correct formation of reproductive organs is critical for successful seedset and perpetuation of the species. Plants have evolved different molecular mechanisms to coordinate flower and seed development at the proper time of the year. Among the plant-specific RELATED TO ABI3 AND VP1 (RAV) family of transcription factors, only TEMPRANILLO1 (TEM1) and TEM2 have been shown to affect reproductive development in Arabidopsis (Arabidopsis thaliana). They negatively regulate the floral transition through direct repression of FLOWERING LOCUS T and GIBBERELLIN 3-OXIDASE1/2, encoding major components of the florigen. Here we identify RAV genes from rice (Oryza sativa), and unravel their regulatory roles in key steps of reproductive development. Our data strongly suggest that, like TEMs, OsRAV9/OsTEM1 has a conserved function as a repressor of photoperiodic flowering upstream of the floral activators OsMADS14 and Hd3a, through a mechanism reminiscent of that one underlying floral transition in temperate cereals. Furthermore, OsRAV11 and OsRAV12 may have acquired a new function in the differentiation of the carpel and the control of seed size, acting downstream of floral homeotic factors. Alternatively, this function may have been lost in Arabidopsis. Our data reveal conservation of RAV gene function in the regulation of flowering time in monocotyledonous and dicotyledonous plants, but also unveil roles in the development of rice gynoecium.

Transcriptome analysis reveals rice MADS13 as an important repressor of the carpel development pathway in ovules.

In angiosperms, floral homeotic genes encoding MADS-domain transcription factors regulate the development of floral organs. Specifically, members of the SEPALLATA (SEP) and AGAMOUS (AG) subfamilies form higher-order protein complexes to control floral meristem determinacy and to specify the identity of female reproductive organs. In rice, the AG subfamily gene OsMADS13 is intimately involved in the determination of ovule identity, since knock-out mutant plants develop carpel-like structures in place of ovules, resulting in female sterility. Little is known about the regulatory pathways at the base of rice gynoecium development. To investigate molecular mechanisms acting downstream of OsMADS13, we obtained transcriptomes of immature inflorescences from wild-type and Osmads13 mutant plants. Among a total of 476 differentially expressed genes (DEGs), a substantial overlap with DEGs from the SEP-family Osmads1 mutant was found, suggesting that OsMADS1 and OsMADS13 may act on a common set of target genes. Expression studies and preliminary analyses of two up-regulated genes encoding Zinc-finger transcription factors indicated that our dataset represents a valuable resource for the identification of both OsMADS13 target genes and novel players in rice ovule development. Taken together, our study suggests that OsMADS13 is an important repressor of the carpel pathway during ovule development.

Gene expression underlying floral epidermal specialization in Aristolochia fimbriata (Aristolochiaceae).

BACKGROUND AND AIMS: The epidermis constitutes the outermost tissue of the plant body. Although it plays major structural, physiological and ecological roles in embryophytes, the molecular mechanisms controlling epidermal cell fate, differentiation and trichome development have been scarcely studied across angiosperms, and remain almost unexplored in floral organs. METHODS: In this study, we assess the spatio-temporal expression patterns of GL2, GL3, TTG1, TRY, MYB5, MYB6, HDG2, MYB106-like, WIN1 and RAV1-like homologues in the magnoliid Aristolochia fimbriata (Aristolochiaceae) by using comparative RNA-sequencing and in situ hybridization assays. KEY RESULTS: Genes involved in Aristolochia fimbriata trichome development vary depending on the organ where they are formed. Stem, leaf and pedicel trichomes recruit most of the transcription factors (TFs) described above. Conversely, floral trichomes only use a small subset of genes including AfimGL2, AfimRAV1-like, AfimWIN1, AfimMYB106-like and AfimHDG2. The remaining TFs, AfimTTG1, AfimGL3, AfimTRY, AfimMYB5 and AfimMYB6, are restricted to the abaxial (outer) and the adaxial (inner) pavement epidermal cells. CONCLUSIONS: We re-evaluate the core genetic network shaping trichome fate in flowers of an early-divergent angiosperm lineage and show a morphologically diverse output with a simpler genetic mechanism in place when compared to the models Arabidopsis thaliana and Cucumis sativus. In turn, our results strongly suggest that the canonical trichome gene expression appears to be more conserved in vegetative than in floral tissues across angiosperms.

TEMPRANILLO is a direct repressor of the microRNA miR172.

In the age-dependent pathway, microRNA 156 (miR156) is essential for the correct timing of developmental transitions. miR156 negatively regulates several SPL genes, which promote the juvenile-to-adult and floral transitions in part through upregulation of miR172. The transcriptional repressors TEMPRANILLO1 (TEM1) and TEM2 delay flowering in Arabidopsis thaliana at least through direct repression of FLOWERING LOCUS T (FT) and gibberellin biosynthetic genes, and have also been reported to participate in the length of the juvenile phase. Levels of TEM mRNA and miR156 decrease gradually, allowing progression through developmental phases. Given these similarities, we hypothesized that TEMs and the miR156/SPL/miR172 module could act through a common genetic pathway. We analyzed the effect of TEMs on levels of miR156, SPL and miR172, tested binding of TEMs to these genes using chromatin immunoprecipitation and analyzed the genetic interaction between TEMs and miR172. We found that TEMs played a stronger role in the floral transition than in the juvenile-to-adult transition. TEM1 repressed MIR172A, MIR172B and MIR172C expressions and bound in vivo to at least MIR172C sequences. Genetic analyses indicated that TEMs affect the regulation of developmental timing through miR172.

AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana.

The effective anti-malarial drug artemisinin (AN) isolated from Artemisia annua is relatively expensive due to the low AN content in the plant as AN is only synthesized within the glandular trichomes. Therefore, genetic engineering of A. annua is one of the most promising approaches for improving the yield of AN. In this work, the AaMYB1 transcription factor has been identified and characterized. When AaMYB1 is overexpressed in A. annua, either exclusively in trichomes or in the whole plant, essential AN biosynthetic genes are also overexpressed and consequently the amount of AN is significantly increased. Artemisia AaMYB1 constitutively overexpressing plants displayed a greater number of trichomes. In order to study the role of AaMYB1 on trichome development and other possibly connected biological processes, AaMYB1 was overexpressed in Arabidopsis thaliana. To support our findings in Arabidopsis thaliana, an AaMYB1 orthologue from this model plant, AtMYB61, was identified and atmyb61 mutants characterized. Both AaMYB1 and AtMYB61 affected trichome initiation, root development and stomatal aperture in A. thaliana. Molecular analyses indicated that two crucial trichome activator genes are misexpressed in atmyb61 mutant plants and in plants overexpressing AaMYB1. Furthermore, AaMYB1 and AtMYB61 are also essential for gibberellin (GA) biosynthesis and degradation in both species by positively affecting the expression of the enzymes that convert GA9 into the bioactive GA4 as well as the enzymes involved in the degradation of GA4 . Overall, these results identify AaMYB1/AtMYB61 as a key component of the molecular network that connects important biosynthetic processes, and reveal its potential value for AN production through genetic engineering.

The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression.

Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning.

TEMPRANILLO Reveals the Mesophyll as Crucial for Epidermal Trichome Formation.

Plant trichomes are defensive specialized epidermal cells. In all accepted models, the epidermis is the layer involved in trichome formation, a process controlled by gibberellins (GAs) in Arabidopsis rosette leaves. Indeed, GA activates a genetic cascade in the epidermis for trichome initiation. Here we report that TEMPRANILLO (TEM) genes negatively control trichome initiation not only from the epidermis but also from the leaf layer underneath the epidermis, the mesophyll. Plants over-expressing or reducing TEM specifically in the mesophyll, display lower or higher trichome numbers, respectively. We surprisingly found that fluorescently labeled GA3 accumulates exclusively in the mesophyll of leaves, but not in the epidermis, and that TEM reduces its accumulation and the expression of several newly identified GA transporters. This strongly suggests that TEM plays an essential role, not only in GA biosynthesis, but also in regulating GA distribution in the mesophyll, which in turn directs epidermal trichome formation. Moreover, we show that TEM also acts as a link between GA and cytokinin signaling in the epidermis by negatively regulating downstream genes of both trichome formation pathways. Overall, these results call for a re-evaluation of the present theories of trichome formation as they reveal mesophyll essential during epidermal trichome initiation.

SHORT VEGETATIVE PHASE Up-Regulates TEMPRANILLO2 Floral Repressor at Low Ambient Temperatures.

Plants integrate day length and ambient temperature to determine the optimal timing for developmental transitions. In Arabidopsis (Arabidopsis thaliana), the floral integrator FLOWERING LOCUS T (FT) and its closest homolog TWIN SISTER OF FT promote flowering in response to their activator CONSTANS under long-day inductive conditions. Low ambient temperature (16°C) delays flowering, even under inductive photoperiods, through repression of FT, revealing the importance of floral repressors acting at low temperatures. Previously, we have reported that the floral repressors TEMPRANILLO (TEM; TEM1 and TEM2) control flowering time through direct regulation of FT at 22°C. Here, we show that tem mutants are less sensitive than the wild type to changes in ambient growth temperature, indicating that TEM genes may play a role in floral repression at 16°C. Moreover, we have found that TEM2 directly represses the expression of FT and TWIN SISTER OF FT at 16°C. In addition, the floral repressor SHORT VEGETATIVE PHASE (SVP) directly regulates TEM2 but not TEM1 expression at 16°C. Flowering time analyses of svp tem mutants indicate that TEM may act in the same genetic pathway as SVP to repress flowering at 22°C but that SVP and TEM are partially independent at 16°C. Thus, TEM2 partially mediates the temperature-dependent function of SVP at low temperatures. Taken together, our results indicate that TEM genes are also able to repress flowering at low ambient temperatures under inductive long-day conditions.

Flowering and trichome development share hormonal and transcription factor regulation.

Gibberellins (GAs) and cytokinins (CKs) are plant hormones that act either synergistically or antagonistically during the regulation of different developmental processes. In Arabidopsis thaliana, GAs and CKs overlap in the positive regulation of processes such as the transition from the vegetative to the reproductive phase and the development of epidermal adaxial trichomes. Despite the fact that both developmental processes originate in the rosette leaves, they occur separately in time and space. Here we review how, as genetic and molecular mechanisms are being unraveled, both processes might be closely related. Additionally, this shared genetic network is not only dependent on GA and CK hormone signaling but is also strictly controlled by specific clades of transcription factor families. Some key flowering genes also control other rosette leaf developmental processes such as adaxial trichome formation. Conversely, most of the trichome activator genes, which belong to the MYB, bHLH and C2H2 families, were found to positively control the floral transition. Furthermore, three MADS floral organ identity genes, which are able to convert leaves into floral structures, are also able to induce trichome proliferation in the flower. These data lead us to propose that the spatio-temporal regulation and integration of diverse signals control different developmental processes, such as floral induction and trichome formation, which are intimately connected through similar genetic pathways.

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.

RAV genes: regulation of floral induction and beyond.

BACKGROUND: Transcription factors of the RAV (RELATED TO ABI3 AND VP1) family are plant-specific and possess two DNA-binding domains. In Arabidopsis thaliana, the family comprises six members, including TEMPRANILLO 1 (TEM1) and TEM2. Arabidopsis RAV1 and TEM1 have been shown to bind bipartite DNA sequences, with the consensus motif C(A/C/G)ACA(N)2-8(C/A/T)ACCTG. Through direct binding to DNA, RAV proteins act as transcriptional repressors, probably in complexes with other co-repressors. SCOPE AND CONCLUSIONS: In this review, a summary is given of current knowledge of the regulation and function of RAV genes in diverse plant species, paying particular attention to their roles in the control of flowering in arabidopsis. TEM1 and TEM2 delay flowering by repressing the production of two florigenic molecules, FLOWERING LOCUS T (FT) and gibberellins. In this way, TEM1 and TEM2 prevent precocious flowering and postpone floral induction until the plant has accumulated enough reserves or has reached a growth stage that ensures survival of the progeny. Recent results indicate that TEM1 and TEM2 are regulated by genes acting in several flowering pathways, suggesting that TEMs may integrate information from diverse pathways. However, flowering is not the only process controlled by RAV proteins. Family members are involved in other aspects of plant development, such as bud outgrowth in trees and leaf senescence, and possibly in general growth regulation. In addition, they respond to pathogen infections and abiotic stresses, including cold, dehydration, high salinity and osmotic stress.

TEMPRANILLO homologs in apple regulate flowering time in the woodland strawberry Fragaria vesca.

The long juvenile period of fruit trees makes their breeding costly and time-consuming. Therefore, flowering time engineering and shortening the juvenile phase have become a breeding priority for the genetic improvement of fruit tree crops. Many economically valuable fruit trees belong to the Rosaceae family including apples and strawberries. TEMPRANILLO (TEM) acts as a key player in flowering time control through inhibiting FT function. Two genes with high sequence similarity with the Arabidopsis TEM genes were isolated from apple (Malus domestica). Due to the complexity of carrying out functional studies in apple, we characterized their function in woodland strawberry as well as their expression in apple. The expression of MdTEM genes in apple tissues from juvenile plants was dramatically higher than that in the tissues from adult trees. In woodland strawberry, the overexpression of MdTEM genes down-regulated FvFT1, FvGA3OX1, and FvGA3OX2 genes in strawberry. The MdTEM-overexpressing lines exhibited delayed flowering, in terms of days to flowering and the number of leaves at flowering. While, RNAi-mediated silencing of TEM resulted in five days earlier flowering, with a lower number of leaves, a higher trichome density, and in some cases, caused in vitro flowering. According to these results and in silico analyses, it can be concluded that MdTEM1 and MdTEM2 can be considered as orthologs of FvTEM and probably AtTEM genes, which play an important role in regulating the juvenile phase and flowering time through regulating FT and GA biosynthetic pathway.

The balance between CONSTANS and TEMPRANILLO activities determines FT expression to trigger flowering.

Seasonal changes in day length influence flowering time in many plant species. In Arabidopsis, flowering is accelerated by exposure to long day (LD). Those inductive photoperiods are perceived in leaves [1] and initiate a long-distance signaling mediated by CO and FT. CO is expressed in the phloem according to a circadian rhythm [2-4]. Only under LD does CO induce FT expression as high levels of CO in the evening coincide with the external light that stabilizes CO protein [4, 5]. Subsequently, FT protein travels through the phloem to the shoot apex where, together with FD, it initiates flowering [6-12]. Despite the photoperiodic induction, a mechanism of floral repression is needed to avoid precocious flowering. We show that TEMPRANILLO genes (TEM1 and TEM2) act as novel direct FT repressors. Molecular and genetic analyses suggest that a quantitative balance between the activator CO and the repressor TEM determines FT levels. Moreover, developmental TEM downregulation marks the timing of flowering, as it shifts the CO/TEM balance in favor of CO activity, allowing FT transcript to reach the threshold level required to trigger flowering. We envision that this might be a general mechanism between long-day plants to ensure a tight regulation of flowering time.

Photoperiod Control of Plant Growth: Flowering Time Genes Beyond Flowering.

Fluctuations in environmental conditions greatly influence life on earth. Plants, as sessile organisms, have developed molecular mechanisms to adapt their development to changes in daylength, or photoperiod. One of the first plant features that comes to mind as affected by the duration of the day is flowering time; we all bring up a clear image of spring blossom. However, for many plants flowering happens at other times of the year, and many other developmental aspects are also affected by changes in daylength, which range from hypocotyl elongation in Arabidopsis thaliana to tuberization in potato or autumn growth cessation in trees. Strikingly, many of the processes affected by photoperiod employ similar gene networks to respond to changes in the length of light/dark cycles. In this review, we have focused on developmental processes affected by photoperiod that share similar genes and gene regulatory networks.

The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity.

The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse plant species. Recent studies in Arabidopsis thaliana have shown that three closely related MADS-box genes, SEPALLATA1 (SEP1), SEP2 and SEP3, are required to specify petals, stamens, and carpels because these organs are converted into sepals in sep1 sep2 sep3 triple mutants. Additional studies indicate that the SEP proteins form multimeric complexes with the products of the B and C organ identity genes. Here, we characterize the SEP4 gene, which shares extensive sequence similarity to and an overlapping expression pattern with the other SEP genes. Although sep4 single mutants display a phenotype similar to that of wild-type plants, we find that floral organs are converted into leaf-like organs in sep1 sep2 sep3 sep4 quadruple mutants, indicating the involvement of all four SEP genes in the development of sepals. We also find that SEP4 contributes to the development of petals, stamens, and carpels in addition to sepals and that it plays an important role in meristem identity. These and other data demonstrate that the SEP genes play central roles in flower meristem identity and organ identity.

Flower and fruit development in Arabidopsis thaliana.

The study of flower development has experienced great advances over the last 15 years. The most important landmark was the proposal of the ABC model in which three different functions of overlapping activities account for the development of the four rings of organs of the eudicot flower. Most interestingly, during recent years this simple and elegant model has been broadly accepted and is applicable to a wide range of plant species. However, recent advances in the characterization of protein interactions and the discovery of the SEPALLATA genes that are required for proper floral organ development have led to a revision of the ABC model. The largely accepted floral quartet model, which includes the new SEPALLATA function, postulates that the development of a specific floral organ is achieved by the formation of a single complex of different MADS-box proteins. The ultimate fate of the flower is to become a fruit, ensuring dispersal of the seeds and therefore survival of the species. The Arabidopsis fruit is a silique or pod. Only in the last five years important advances have been made in establishing the differentiation of the tissues required for the opening of the fruit: the valve margins and dehiscence zone. Classical genetic analyses and molecular biology approaches have pointed to the involvement of the transcription factors SHP, ALC and IND in the formation of these tissues and of FUL and RPL in repressing this identity in the bordering tissues, valves and replum, respectively.