Axillary bud development in pineapple nodal segments correlates with changes on cell cycle gene expression, hormone level, and sucrose and glutamate contents

During the process of lateral organ development after plant decapitation, cell division and differentiation occur in a balanced manner initiated by specific signaling, which triggers the reentrance into the cell cycle. Here, we investigated short-term variations in the content of some endogenous signals, such as auxin, cytokinins (Cks), and other mitogenic stimuli (sucrose and glutamate), which are likely correlated with the cell cycle reactivation in the axillary bud primordium of pineapple nodal segments. Transcript levels of cell cycle-associated genes, CycD2;1, and histone H2A were analyzed. Nodal segments containing the quiescent axillary meristem cells were cultivated in vitro during 24 h after the apex removal and de-rooting. From the moment of stem apex and root removal, decapitated nodal segment (DNS) explants showed a lower indol-3-acetic acid (IAA) concentration than control explants, and soon after, an increase of endogenous sucrose and iP-type Cks were detected. The decrease of IAA may be the primary signal for cell cycle control early in G1 phase, leading to the upregulation of CycD2;1 gene in the first h. Later, the iP-type Cks and sucrose could have triggered the progression to S-phase since there was an increase in H2A expression at the eighth h. DNS explants revealed substantial increase in Z-type Cks and glutamate from the 12th h, suggesting that these mitogens could also operate in promoting pineapple cell cycle progression. We emphasize that the use of non-synchronized tissue rather than synchronous cell suspension culture makes it more difficult to interpret the results of a dynamic cell division process. However, pineapple nodal segments cultivated in vitro may serve as an interesting model to shed light on apical dominance release and the reentrance of quiescent axillary meristem cells into the cell cycle.

Global analysis of the sugarcane microtranscriptome reveals a unique composition of small RNAs associated with axillary bud outgrowth

Axillary bud outgrowth determines shoot architecture and is under the control of endogenous hormones and a fine-tuned gene-expression network, which probably includes small RNAs (sRNAs). Although it is well known that sRNAs act broadly in plant development, our understanding about their roles in vegetative bud outgrowth remains limited. Moreover, the expression profiles of microRNAs (miRNAs) and their targets within axillary buds are largely unknown. Here, we employed sRNA next-generation sequencing as well as computational and gene-expression analysis to identify and quantify sRNAs and their targets in vegetative axillary buds of the biofuel crop sugarcane (Saccharum spp.). Computational analysis allowed the identification of 26 conserved miRNA families and two putative novel miRNAs, as well as a number of trans-acting small interfering RNAs. sRNAs associated with transposable elements and protein-encoding genes were similarly represented in both inactive and developing bud libraries. Conversely, sequencing and quantitative reverse transcription-PCR results revealed that specific miRNAs were differentially expressed in developing buds, and some correlated negatively with the expression of their targets at specific stages of axillary bud development. For instance, the expression patterns of miR159 and its target GAMYB suggested that they may play roles in regulating abscisic acid-signalling pathways during sugarcane bud outgrowth. Our work reveals, for the first time, differences in the composition and expression profiles of diverse sRNAs and targets between inactive and developing vegetative buds that, together with the endogenous balance of specific hormones, may be important in regulating axillary bud outgrowth. © 2013 © The Author(2) [2013].

Axillary bud outgrowth: sending a message

Mutants that branch profusely in the presence of a growing shoot tip have highlighted the role of graft-transmissible signals that are produced in roots and stem. Orthologous genes in Arabidopsis, pea and petunia are involved in the transmission of a novel long-distance message. These genes show varying degrees of regulation by auxin and an auxin-independent feedback system, and encode enzymes that might act on carotenoid-like substrates. Axillary bud outgrowth is under homeostatic control, involving developmental stages or checkpoints. Perturbation of the long-range messaging and auxin depletion does not guarantee that bud outgrowth will ensue at a particular node.

Auxin dynamics after decapitation are not correlated with the initial growth of axillary buds

One of the first and most enduring roles identified for the plant hormone auxin is the mediation of apical dominance. Many reports have claimed that reduced stem indole-3-acetic acid (IAA) levels and/ or reduced basipetal IAA transport directly or indirectly initiate bud growth in decapitated plants. We have tested whether auxin inhibits the initial stage of bud release, or subsequent stages, in garden pea (Pisum sativum) by providing a rigorous examination of the dynamics of auxin level, auxin transport, and axillary bud growth. We demonstrate that after decapitation, initial bud growth occurs prior to changes in IAA level or transport in surrounding stem tissue and is not prevented by an acropetal supply of exogenous auxin. We also show that auxin transport inhibitors cause a similar auxin depletion as decapitation, but do not stimulate bud growth within our experimental time- frame. These results indicate that decapitation may trigger initial bud growth via an auxin-independent mechanism. We propose that auxin operates after this initial stage, mediating apical dominance via autoregulation of buds that are already in transition toward sustained growth.

Regulation of strawberry growth and development

Strawberries (Fragaria sp.) are adapted to diverse environmental conditions from the tropics to about 70ºN, so different responses to environmental conditions can be found. Most genotypes of garden strawberry (F. x ananassa Duch.) and woodland strawberry (F. vesca L.) are short-day (SD) plants that are induced to flowering by photoperiods under a critical limit, but also various photoperiod x temperature interactions can be found. In addition, continuously flowering everbearing (EB) genotypes are found. In addition to flowering, axillary bud differentiation in strawberry is regulated by photoperiod. In SD conditions, axillary buds differentiate to rosette-like structures called "branch crowns", whereas in long-day conditions (LD) they form runners, branches with 2 long internodes followed by a daughter plant (leaf rosette). The number of crown branches determines the yield of the plant, since inflorescences are formed from the apical meristems of the crown. Although axillary bud differentiation is an important developmental process in strawberries, its environmental and hormonal regulation has not been characterized in detail. Moreover, the genetic mechanisms underlying axillary bud differentiation and regulation of flowering time in these species are almost completely unresolved. These topics have been studied in this thesis in order to enhance strawberry research, cultivation and breeding. The results showed that 8-12 SD cycles suppressed runner initiation from the axillary buds of the garden strawberry cv. Korona with the concomitant induction of crown branching, and 3 weeks of SD was sufficient for the induction of flowering in the main crown. Furthermore, a second SD treatment given a few weeks after the first SD period can be used to induce flowering in the primary branch crowns and to induce the formation of secondary branches. Thus, artificial SD treatments effectively stimulate crown branching, providing one means for the increase of cropping (yield) potential in strawberry. It was also shown by growth regulation applications, quantitave hormone analysis and gene expression analysis that gibberellin (GA) is one of the key signals involved in the photoperiod control of shoot differentiation. The results indicate that photoperiod controls GA activity specifically in axillary buds, thereby determining bud fate. It was further shown that chemical control of GA biosynthesis by prohexadione-calcium can be utilized to prevent excessive runner formation and induce crown branching in strawberry fields. Moreover, ProCa increased berry yield up to 50%, showing that it is an easier and more applicable alternative to artificial SD treatments for controlling strawberry crown development and yield. Finally, flowering gene pathways in Fragaria were explored by searching for homologs of 118 Arabidopsis thaliana flowering-time genes. In total, 66 gene homologs were identified, and they distributed to all known flowering pathways, suggesting the presence of these pathways also in strawberry. Expression analysis of selected genes revealed that the mRNA of putative floral identity gene APETALA1 accumulated in the shoot apex of the EB genotype after the induction of flowering, whereas it was absent in vegetative SD genotype, indicating the usefulness of this gene product as the marker of floral initiation. The present data enables the further exploration of strawberry flowering pathways with genetic transformation, gene mapping and transcriptomics methods.

In vitro plant regeneration from different seedling explants of blackgram [Vigna mungo (L.) Hepper] via organogenesis

Callus induction and morphogenesis from different blackgram explants were tested on MS basal medium supplemented with B5 vitamins, IAA, NAA, IBA, KIN and BAP individually and in combinations. The explants were hypocotyl, epicotyl, axillary bud, cotyledonary node and immature leaf. The optimal levels of the frequency of callus induction was 22.8 mu M of IAA or 16.1 mu M NAA and in combination with 2.2 mu M of BAP. Among the seedling explants, hypocotyl was found to be more efficient in producing callus. Shoots mere induced from callus cultures of hypocotyls, epicotyls, axillary bud, cotyledonary node and immature leaf with varying frequencies in the medium containing KIN (2.3-9.3 mu M) or BAP (2.2-8.8 mu M) and in combination with IAA (2.8 mu M) or NAA (2.6 mu M). Multiple shoots were obtained using cotyledonary node segments. The regenerated shoots rooted best on MS basal medium containing 9.8 mu M IBA. Seventy three per cent of the shoots produced roots, and 80-85% of the plantlets survived under greenhouse condition.

Estratégias para a valorização do coberto vegetal da Ilha de Porto Santo

No presente trabalho desenvolveram-se estudos visando a valorização do coberto vegetal da Ilha de Porto Santo, através de duas metodologias de investigação complementares: a) preservação e reintrodução na Ilha de uma espécie endémica e em risco do Arquipélago da Madeira (Olea maderensis) e de uma espécie naturalizada (Olea europaea ssp. europaea var. sylvestris), recorrendo para o efeito a técnicas de biotecnologia (micropropagação); b) análise da percepção da comunidade local e visitante sobre o fenómeno da desertificação e a valorização do coberto vegetal bem como a sua aceitação relativamente à aplicação de técnicas de biotecnologia (para micropropagar e reintroduzir espécies de oliveira na Ilha) para minimização do processo de desertificação. A dissertação estrutura-se em quatro partes principais. A Parte I caracteriza a Ilha de Porto Santo em termos geográficos, geológicos, climáticos, sócio-economicos e do uso do solo. Enquadra, ainda, o problema da desertificação, através da caracterização/evolução do coberto vegetal ao longo dos anos. Finalmente apresentam-se os objectivos gerais deste estudo. A Parte II centra-se no desenvolvimento de metodologias no âmbito da biotecnologia vegetal para propagação de espécies de O. maderensis e O. europaea ssp. europaea var. sylvestris. em larga escala. Esta parte está dividida em seis capítulos. O Capítulo II.1 aborda a distribuição geográfica das espécies de oliveira e faz uma revisão bibliográfica dos aspectos mais importantes da micropropagação de oliveira (O. europaea L.), principalmente através da micropropagação por estimulação de gomos axilares. No final deste capítulo apresentam-se os objectivos específicos desta investigação. No Capítulo II.2 faz-se a caracterização genética de genótipos de O. maderensis do Arquipélago da Madeira através da análise da ploidia e do conteúdo em DNA por citometria de fluxo (FCM) e através da detecção de polimorfismos por análise de microssatélites (SSR). Nesta análise usaram-se ainda outros genótipos, nomeadamente: O. europaea ssp. europaea var. sylvestris, O. cerasiformes e O. europaea ssp. europaea var. europaea. Este estudo contribuiu para uma melhor caracterização desta espécie e permitiu a detecção de um nível de ploidia novo no género Olea (tetraploidia). O Capítulo II.3 descreve a optimização das condições de cultura in vitro (e.g. desinfecção, meio de cultura e enraizamento) para propagar e preservar a O. maderensis. Avalia-se ainda a “performance” dos rebentos in vitro (taxas de crescimento, avaliação da aparência das folhas e estudos fisiológicos), de modo a confirmar a optimização das condições de propagação. Neste capítulo define-se um meio novo (OMG) para propagação desta espécie endémica. O Capítulo II.4 descreve dois protocolos de micropropagação e aclimatização de ambas as espécies (O. maderensis e O. europaea ssp. europaea var. sylvestris) e a qualidade das plantas (“true-to-type”) é avaliada através da possível ocorrência de variabilidade genética através de FCM (ploidia) e SSRs. O Capítulo II.5 descreve um protocolo eficiente de aclimatização ao campo de O. maderensis e avalia a “performance” das plantas micropropagadas no campo através da análise de parâmetros fisiológicos durante o processo. O Capítulo II.6 apresenta os estudos em curso relativamente às plantas de O. maderensis em aclimatização no campo, bem como a introdução de plantas micropropagadas num outro local da Ilha com um maior grau de degradação dos solos. Estas estratégias estão a ser aplicadas juntamente com a DRFRAM, no âmbito de programas de florestação em curso. Finalmente é realçada a necessidade de estudos semelhantes com outras espécies nativas. Na Parte III, são apresentados os estudos sobre a percepção da comunidade local relativamente à valorização do coberto vegetal para a minimização dos processos de degradação dos solos/desertificação. A introdução faz o enquadramento teórico sobre o fenómeno da desertificação, particularmente na Ilha de Porto Santo e sobre a percepção social da desertificação. São ainda apresentados os objectivos específicos desta investigação. A metodologia adoptada recorreu à aplicação de inquéritos por questionário à população residente e aos visitantes da Ilha de Porto Santo e ainda a realização de inquéritos por entrevista a algumas entidades e especialistas. Estes estudos permitiram verificar que existe uma nítida consciência da situação de risco da ilha, das medidas tomadas e a tomar e da premência da resolução do problema. Face ao recurso de estratégias alternativas envolvendo biotecnologia, e apesar de existir algum desconhecimento, concluiu-se ainda que a população manifesta aceitação, desde que essas estratégias valorizem o coberto vegetal e, assim, ajudem a combater a degradação biofísica dos solos. Finalmente são apresentadas as conclusões e algumas recomendações. Na Parte IV apresentam-se as conclusões gerais e perspectivas futuras, onde o potencial ambiental destas oliveiras bravas micropropagadas é destacado, bem como é considerado o alargamento da aplicação destas estratégias a outras espécies indígenas em risco, nesta Ilha (e noutros locais). São ainda resumidas as principais visões da população e das entidades e dos especialistas que poderão contribuir para apoiar a elaboração de medidas de mitigação e prevenção no combate ao processo de degradação dos solos/desertificação em curso.

Atividade da enzima bromelina emplantas de abacaxi (Ananas comosus L. Merril), sob condições de salinidade in vitro

The objectives of this work were to characterize pineapple plants (Ananas comosus L. Merril) cv. Smooth Cayenne, cultured in vitro, in saline medium, in relation to bromelian activity, identifying the parts of the plant with the highest bromelian activity. Also under aim was the study of the influence of saline stress on the enzyme activity. Axillary buds of pineapple were cultivated in vitro in MS medium, supplemented with 2 mg.L-1 BAP and 1 mg.L-1 NAA. The levels of salinity tested were: 0.57 g.L-1 NaCl, 1.15 g.L-1 NaCl, and 2.30 g.L-1 NaCl. Bromelian activity was evaluated in the development of buds, shoots, and roots. The results showed that bromelian activity was higher in buds at the highest salt concentration at 15 days. Cultured shoots showed bromelian activity decreasing in the saline treatments in all the collection, up to 60 days in culture. The roots showed higher bromelian activity in the roots in saline medium.

Análises bioquímicas e hsitológicas na micropropagação de abacaxizeiro 'Gomo de Mel' submetido a reguladores vegetais

Pós-graduação em Agronomia (Horticultura) - FCA

Branching genes are conserved across species. Genes controlling a novel signal in pea are coregulated by other long-distance signals

Physiological and genetic studies with the ramosus (rms) mutants in garden pea (Pisum sativum) and more axillary shoots (max) mutants in Arabidopsis (Arabidopsis thaliana) have shown that shoot branching is regulated by a network of long-distance signals. Orthologous genes RMS1 and MAX4 control the synthesis of a novel graft-transmissible branching signal that may be a carotenoid derivative and acts as a branching inhibitor. In this study, we demonstrate further conservation of the branching control system by showing that MAX2 and MAX3 are orthologous to RMS4 and RMS5, respectively. This is consistent with the longstanding hypothesis that branching in pea is regulated by a novel long-distance signal produced by RMS1 and RMS5 and that RMS4 is implicated in the response to this signal. We examine RMS5 expression and show that it is more highly expressed relative to RMS1, but under similar transcriptional regulation as RMS1. Further expression studies support the hypothesis that RMS4 functions in shoot and rootstock and participates in the feedback regulation of RMS1 and RMS5 expression. This feedback involves a second novel long-distance signal that is lacking in rms2 mutants. RMS1 and RMS5 are also independently regulated by indole-3-acetic acid. RMS1, rather than RMS5, appears to be a key regulator of the branching inhibitor. This study presents new interactions between RMS genes and provides further evidence toward the ongoing elucidation of a model of axillary bud outgrowth in pea.

Modeling Nymphoides architecture: A morphological analysis of Nymphoides aquatica (Menyanthaceae)

• Premise of the study: Species in the aquatic genus Nymphoides have inflorescences that appear to arise from the petioles of floating leaves. The inflorescence-floating leaf complex can produce vegetative propagules and/or additional inflorescences and leaves. We analyzed the morphology of N. aquatica to determine how this complex relates to whole plant architecture and whether whole plant growth is sympodial or monopodial. • Methods: We used dissections, measurements, and microscopic observations of field-collected plants and plants cultivated for 2 years in outdoor tanks in south Florida, USA. • Key results: Nymphoides aquatica had a submerged plagiotropic rhizome that produced floating leaves in an alternate/spiral phyllotaxy. Rhizomes were composed of successive sympodial units that varied in the number of leaves produced before the apex terminated. The basic sympodial unit had a prophyll that subtended a renewal-shoot bud, a short-petioled leaf (SPL) with floating lamina, and an inflorescence; the SPL axillary bud expanded as a vegetative propagule. Plants produced either successive basic sympodial units or expanded sympodia that intercalated long-petioled leaves between the prophyll and the SPL. • Conclusions: Nymphoides aquatica grows sympodially, forming a rhizome composed of successive basic sympodia and expanded sympodial units. Variations on these types of sympodial growth help explain the branching patterns and leaf morphologies described for other Nymphoides species. Monitoring how these two sympodial phases are affected by water depth provides an ecologically meaningful way to assess N. aquatica’s responses to altered hydrology.

Functions for rice RFL in vegetative axillary meristem specification and outgrowth

Roles for the transcription factor RFL in rice axillary meristem development were studied. Its regulatory effects on LAX1, CUC1, and OsPIN3 reveal its functions in axillary meristem specification and outgrowth.Axillary meristems (AMs) are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers, and tillering mutants reveal an interplay between transcription factors and the phytohormones auxin and strigolactone as some factors that underpin this developmental process. Previous studies showed that knockdown of the transcription factor gene RFL reduced tillering and caused a very large decrease in panicle branching. Here, the relationship between RFL, AM initiation, and outgrowth was examined. We show that RFL promotes AM specification through its effects on LAX1 and CUC genes, as their expression was modulated on RFL knockdown, on induction of RFL:GR fusion protein, and by a repressive RFL-EAR fusion protein. Further, we report reduced expression of auxin transporter genes OsPIN1 and OsPIN3 in the culm of RFL knockdown transgenic plants. Additionally, subtle change in the spatial pattern of IR4 DR5:GFP auxin reporter was observed, which hints at compromised auxin transport on RFL knockdown. The relationship between RFL, strigolactone signalling, and bud outgrowth was studied by transcript analyses and by the tillering phenotype of transgenic plants knocked down for both RFL and D3. These data suggest indirect RFL-strigolactone links that may affect tillering. Further, we show expression modulation of the auxin transporter gene OsPIN3 upon RFL:GR protein induction and by the repressive RFL-EAR protein. These modified forms of RFL had only indirect effects on OsPIN1. Together, we have found that RFL regulates the LAX1 and CUC genes during AM specification, and positively influences the outgrowth of AMs though its effects on auxin transport.

Bud source, asepsis and benzylaminopurine (BAP) effect on yacon (Polymnia sonchifolia) micropropagation

The yacon (Polymnia sonchifolia) is used largely for the high fructan content of its tubers; consequently, it is a good alternative for diabetics. One of the more important restricting factors of the commercial production of yacon is its susceptibility to nematode attack. This, as well as germplasm bank maintenance, justifies the importance of in vitro propagation of this species. In this way, our work aimed to verify the best asepsis method for yacon for the in vitro establishment from the rhizophore and the axillary buds of the aerial parts, and the effect of benzylaminopurine (BAP) addition to the culture medium. The number of contaminated cultures, the occurrence of phenolic oxidation and the occurrence of a vitreous aspect, showed differences with bud source, immersion time for asepsis, and BAP use. The results contribute to establishing a yacon micro propagation procedure.