Flower Meristem Identity Genes Research Articles

Decoding Gene Expression Signatures Underlying Vegetative to Inflorescence Meristem Transition in the Common Bean

The tropical common bean (Phaseolus vulgaris L.) is an obligatory short-day plant that requires relaxation of the photoperiod to induce flowering. Similar to other crops, photoperiod-induced floral initiation depends on the differentiation and maintenance of meristems. In this study, the global changes in transcript expression profiles were analyzed in two meristematic tissues corresponding to the vegetative and inflorescence meristems of two genotypes with different sensitivities to photoperiods. A total of 3396 differentially expressed genes (DEGs) were identified, and 1271 and 1533 were found to be up-regulated and down-regulated, respectively, whereas 592 genes showed discordant expression patterns between both genotypes. Arabidopsis homologues of DEGs were identified, and most of them were not previously involved in Arabidopsis floral transition, suggesting an evolutionary divergence of the transcriptional regulatory networks of the flowering process of both species. However, some genes belonging to the photoperiod and flower development pathways with evolutionarily conserved transcriptional profiles have been found. In addition, the flower meristem identity genes APETALA1 and LEAFY, as well as CONSTANS-LIKE 5, were identified as markers to distinguish between the vegetative and reproductive stages. Our data also indicated that the down-regulation of the photoperiodic genes seems to be directly associated with promoting floral transition under inductive short-day lengths. These findings provide valuable insight into the molecular factors that underlie meristematic development and contribute to understanding the photoperiod adaptation in the common bean.

A Machine-Learning Analysis of Flowering Gene Expression in the CDC Frontier Chickpea Cultivar

Abstract—We have analyzed the gene expression dynamics in floral transition in the CDC Frontier chickpea cultivar. We provide a model, in several versions, to predict the expression dynamics of five flowering genes, taking the expression of their regulators as an input. The models were trained using the random forest method on the previously published expression data for ten flowering genes under the short- and long-day growing conditions. The resulting models correctly predict the dynamics of the average expression levels under long days. We show that the models for CDC Frontier mainly reproduce the regulatory interactions between the key genes described for the Arabidopsis thaliana model plant. Based on the analysis, we hypothesize that the short-day data and the long-day data contain qualitatively different information, which may be due to different regulatory modules that function in different conditions. For the regulators of the flower meristem identity genes AP1 and LFY, our models predict FTa3 as the main activator and TFL1c as the main repressor under long days.

Molecular cloning and functional analysis of the BplSPL1 gene from Betula platyphylla Suk.

BplSPL1 gene was identified in Betula platyphylla Suk, and its sequence features and gene function expression have provided useful information for further studies on function. SQUAMOSA promoter-binding protein-like (SPL) are plant-specific transcription factors that play an important regulatory role in plant growth and development. In this study, an SPL gene, BplSPL1, was cloned from Betula platyphylla Suk. (white birch). Sequence analysis revealed that BplSPL1 has a 405 bp open reading frame (ORF) and encodes a protein of 134 amino acids, which contains a highly conserved SQUAMOSA promoter-binding protein (SBP) domain belonging to the SPL family. Quantitative RT-PCR analysis showed that BplSPL1 was more highly expressed in mature leaf as well as male and female inflorescences. The ectopic expression of BplSPL1 in Arabidopsis led to early flowering, reduced number of rosette leaves, and increased number of lateral branches. We also found that plant height of the transgenic plants decreased, and length of the siliques was shorter in the 35S::BplSPL1 transgenic plants. Further studies showed that the early-flowering phenotype in 35S::BplSPL1 transgenic plants was related to changes in the expression of flowering time genes and flower meristem identity genes, including CUC, FUL, DCL, and SOC1. Therefore, the above results indicate that: (1) BplSPL1 may be involved in regulating flowering time and phase transition, and (2) BplSPL1 may interact with other genes to regulate plant height and the development of lateral branches and fruit.

Cloning and functional characterization of a novel BpSEP4 gene from Betula platyphylla Suk.

SEPALLATA (SEP) genes, members of a family of MADS-box transcription factors, have essential roles in floral organ development and flowering time. In this study, a novel SEP gene, BpSEP4, was identified from Betula platyphylla Suk. The full-length BpSEP4 cDNA was 928 bp, and it contained highly conserved MADS and K domains characteristic of the SEP subfamily. Subcellular location analysis demonstrated that BpSEP4 localized in the nucleus. qRT-PCR analysis showed that BpSEP4 expression was highest in reproductive organs, where it played an essential role in inflorescence development. The ectopic expression of BpSEP4 in Arabidopsis thaliana caused early flowering and aberrant floral organ development. Moreover, delayed flower senescence and abscission were also observed in transgenic Arabidopsis plants. Further studies demonstrated that the early-flowering phenotype in 35S::BpSEP4 transgenic plants was caused by changes in the expression of flowering time genes and flower meristem identity genes, including CO, FT, SOC1, and TFL1. These results indicate that BpSEP4 is a SEP ortholog that may be involved in controlling flowering time and floral development.

Flowering time and the identification of floral marker genes in Solanum tuberosum ssp. andigena.

Solanaceae is a family of flowering plants that includes agricultural species such as tomato (Solanum lycopersicum), eggplant (S. melongena), pepper (Capsicum annuum), and potato (S. tuberosum). The transition from the vegetative to reproductive stage has been extensively investigated in tomato as it affects fruit yield. While potato has mainly been studied with regards to the formation of storage organs, control of flowering time is a subject of increasing interest as development of true seeds is becoming more important for future breeding strategies. Here, we describe a robust growth regime for synchronized development of S. tuberosum ssp. andigena. Using SEM to analyse the developmental stages of the shoot apical meristem (SAM) throughout the floral transition, we show that andigena is a facultative long-day plant with respect to flowering. In addition, we identify the flower meristem identity gene MACROCALYX (StMC) as a marker to distinguish between the vegetative and reproductive stages. We show that the expression of WUSCHEL HOMEOBOX 9 (StWOX9) and ANANTHA (StAN) are specific to the inflorescence meristem and flower meristems in the cyme, respectively. The expression patterns of homologs of Arabidopsis flowering-time regulators were studied, and indicated that SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (StSOC1) and StFD might regulate flowering similar to other plant species.

Turning Meristems into Fortresses.

TERMINAL FLOWER1 (TFL1) was named from knockout Arabidopsis thaliana mutants in which the inflorescence abnormally terminates into a flower. In wild type plants, the expression of TFL1 in the center of the inflorescence meristem represses the flower meristem identity genes LEAFY (LFY) and APETALA1 (AP1) to maintain indeterminacy. LFY and AP1 are activated by flowering signals that antagonize TFL1. Its characterization in numerous species revealed that the TFL1-mediated regulation of meristem fate has broader impacts on plant development than originally depicted in A. thaliana. By blocking floral transition, TFL1 genes participate in the control of juvenility, shoot growth pattern, inflorescence architecture, and the establishment of life history strategies. Here, we contextualize the role of the TFL1-mediated protection of meristem indeterminacy throughout plant development.

Identification and characterization of FRUITFULL-like genes from Platanus acerifolia, a basal eudicot tree

The function of euAP1 and euFUL in AP1/FUL lineage have been well characterized in core eudicots, and they play common and distinct roles in plant development. However, the evolution and function of FUL-like genes is poorly understood in basal eudicots. In this study, we identified three FUL-like genes PlacFL1/2/3 from London plane (Platanus acerifolia). Sequence alignment and phylogenetic analysis indicated that PlacFL1/2/3 are AP1/FUL orthologs and encoded proteins containing FUL motif and paleoAP1 motif. Quantitative real-time PCR (qRT-PCR) analysis showed that PlacFL1/2/3 were expressed in both vegetative and reproductive tissues, but with distinct spatiotemporal patterns. In contrast to PlacFL1 and PlacFL3, PlacFL2 exhibited higher expression levels and broader expression regions, and that the expression of PlacFL2 gene showed a decreasing and increasing tendency in subpetiolar buds during dormancy induction and breaking, respectively. Overexpression of PlacFLs in Arabidopsis and PlacFL3 in tobacco resulted in early flowering, as well as early termination of inflorescence meristems for transgenic Arabidopsis plants. The expression changes of flowering time and flower meristem identity genes in transgenic Arabidopsis lines with different PlacFLs suggested that PlacFL2 and PlacFL3 may regulate different downstream genes to perform divergent functions. Yeast two-hybrid analysis indicated that PlacFLs interacted strongly with PlacSEP proteins, and PlacFL3 instead of PlacFL1 and PlacFL2 could also form a homodimer and interact with D-class proteins. Our results suggest that PlacFLs may play conserved functions in regulating flowering and flower development, and PlacFL2 might also be involved in dormancy regulation. The research helps us to understand the functional evolution of FUL-like genes in basal eudicots, especially in perennial woody species.

Identification and characterization of the flower meristem identity gene MhyLFY in mycoheterotrophic plant Monotropa hypopitys.

The gene encoding the transcription factor LEAFY was identified in the genome of the mycoheterotrophic plant, pinesap Monotropa hypopitys. In the transcriptomes of roots, bracts, and flowers of flowering pinesaps, the MhyLFY gene expression was absent. These data suggest the conservativeness of the LFY-dependent mechanism of flower meristem identity and flower formation in heterotrophic species with some differences associated to the specificity of development and the structure of such plants. The pinesap flowering under the control of the transcription factor MhyLFY may be initiated either in an embryonic inflorescence during spring dormancy release of adventitious root buds or in an inflorescence of a growing reproductive stem after photoperiodic induction.

Expression patterns of Passiflora edulis APETALA1/FRUITFULL homologues shed light onto tendril and corona identities

BackgroundPassiflora (passionflowers) makes an excellent model for studying plant evolutionary development. They are mostly perennial climbers that display axillary tendrils, which are believed to be modifications of the inflorescence. Passionflowers are also recognized by their unique flower features, such as the extra whorls of floral organs composed of corona filaments and membranes enclosing the nectary. Although some work on Passiflora organ ontogeny has been done, the developmental identity of both Passiflora tendrils and the corona is still controversial. Here, we combined ultrastructural analysis and expression patterns of the flower meristem and floral organ identity genes of the MADS-box AP1/FUL clade to reveal a possible role for these genes in the generation of evolutionary novelties in Passiflora.ResultsWe followed the development of structures arising from the axillary meristem from juvenile to adult phase in P. edulis. We further assessed the expression pattern of P. edulis AP1/FUL homologues (PeAP1 and PeFUL), by RT-qPCR and in situ hybridization in several tissues, correlating it with the developmental stages of P. edulis. PeAP1 is expressed only in the reproductive stage, and it is highly expressed in tendrils and in flower meristems from the onset of their development. PeAP1 is also expressed in sepals, petals and in corona filaments, suggesting a novel role for PeAP1 in floral organ diversification. PeFUL presented a broad expression pattern in both vegetative and reproductive tissues, and it is also expressed in fruits.ConclusionsOur results provide new molecular insights into the morphological diversity in the genus Passiflora. Here, we bring new evidence that tendrils are part of the Passiflora inflorescence. This points to the convergence of similar developmental processes involving the recruitment of genes related to flower identity in the origin of tendrils in different plant families. The data obtained also support the hypothesis that the corona filaments are likely sui generis floral organs. Additionally, we provide an indication that PeFUL acts as a coordinator of passionfruit development.

Evolutionary Co-Option of Floral Meristem Identity Genes for Patterning of the Flower-Like Asteraceae Inflorescence.

The evolutionary success of Asteraceae, the largest family of flowering plants, has been attributed to the unique inflorescence architecture of the family, which superficially resembles an individual flower. Here, we show that Asteraceae inflorescences (flower heads, or capitula) resemble solitary flowers not only morphologically but also at the molecular level. By conducting functional analyses for orthologs of the flower meristem identity genes LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) in Gerbera hybrida, we show that GhUFO is the master regulator of flower meristem identity, while GhLFY has evolved a novel, homeotic function during the evolution of head-like inflorescences. Resembling LFY expression in a single flower meristem, uniform expression of GhLFY in the inflorescence meristem defines the capitulum as a determinate structure that can assume floral fate upon ectopic GhUFO expression. We also show that GhLFY uniquely regulates the ontogeny of outer, expanded ray flowers but not inner, compact disc flowers, indicating that the distinction of different flower types in Asteraceae is connected with their independent evolutionary origins from separate branching systems.

Ectopic expression of Jatropha curcas APETALA1 (JcAP1) caused early flowering in Arabidopsis, but not in Jatropha.

Jatropha curcas is a promising feedstock for biofuel production because Jatropha oil is highly suitable for the production of biodiesel and bio-jet fuels. However, Jatropha exhibits a low seed yield as a result of unreliable and poor flowering. APETALA1 (AP1) is a floral meristem and organ identity gene in higher plants. The flower meristem identity genes of Jatropha have not yet been identified or characterized. To better understand the genetic control of flowering in Jatropha, an AP1 homolog (JcAP1) was isolated from Jatropha. An amino acid sequence analysis of JcAP1 revealed a high similarity to the AP1 proteins of other perennial plants. JcAP1 was expressed in inflorescence buds, flower buds, sepals and petals. The highest expression level was observed during the early developmental stage of the flower buds. The overexpression of JcAP1 using the cauliflower mosaic virus (CaMV) 35S promoter resulted in extremely early flowering and abnormal flowers in transgenic Arabidopsis plants. Several flowering genes downstream of AP1 were up-regulated in the JcAP1-overexpressing transgenic plant lines. Furthermore, JcAP1 overexpression rescued the phenotype caused by the Arabidopsis AP1 loss-of-function mutant ap1-11. Therefore, JcAP1 is an ortholog of AtAP1, which plays a similar role in the regulation of flowering in Arabidopsis. However, the overexpression of JcAP1 in Jatropha using the same promoter resulted in little variation in the flowering time and floral organs, indicating that JcAP1 may be insufficient to regulate flowering by itself in Jatropha. This study helps to elucidate the function of JcAP1 and contributes to the understanding of the molecular mechanisms of flower development in Jatropha.

Capsicum annuum S (CaS) promotes reproductive transition and is required for flower formation in pepper (Capsicum annuum)

The genetic control of the transition to flowering has mainly been studied in model species, while few data are available in crop species such as pepper (Capsicum spp.). To elucidate the genetic control of the transition to flowering in pepper, mutants that lack flowers were isolated and characterized. Genetic mapping and sequencing allowed the identification of the gene disrupted in the mutants. Double mutants and expression analyses were used to characterize the relationships between the mutated gene and other genes controlling the transition to flowering and flower differentiation. The mutants were characterized by a delay in the initiation of sympodial growth, a delay in the termination of sympodial meristems and complete inhibition of flower formation. Capsicum annuum S (CaS), the pepper (Capsicum annuum) ortholog of tomato (Solanum lycopersicum) COMPOUND INFLORESCENCE and petunia (Petunia hybrida) EVERGREEN, was found to govern the mutant phenotype. CaS is required for the activity of the flower meristem identity gene Ca-ANANTHA and does not affect the expression of CaLEAFY. CaS is epistatic over other genes controlling the transition to flowering with respect to flower formation. Comparative homologous mutants in the Solanaceae indicate that CaS has uniquely evolved to have a critical role in flower formation, while its role in meristem maturation is conserved in pepper, tomato and petunia.

Identification and Characterization of PpLFL, a Homolog of FLORICAULA/LEAFY in Peach (Prunus persica)

A LEAFY/FLORICAULA (LFY/FLO) homolog PpLFL (PrunuspersicaLEAFY/FLORICAULALike) gene was isolated from axillary buds of peach (Prunus persica (L.) Batsch. cv. Bayuecui) during flower induction period. The open reading frame of PpLFL spanned 1,248 bp, encoding a putative protein of 415 amino acid residues, which was with high similarity (50.48 %–84.69 %) to other FLO/LFY inferred proteins from different species. The spatial expression patterns of PpLFL were detected in axillary buds during the periods of flower induction by using immunohistolocalisation. The results showed that PpLFL gene was mainly expressed during flower induction time, and also in leaf and petal promordia at the SAM. For further functional analysis, the PpLFL was constitutively expressed in the Arabidopsis lfy mutant background, and the results showed that overexpression of PpLFL under the control of CaMV 35S promoter can accelerate flowering and give rise to normal flower organs. Our results suggest that PpLFL might play an important role in flower induction, and could act as a functional flower meristem identity gene in peach.

Integrating long‐day flowering signals: a LEAFY binding site is essential for proper photoperiodic activation of APETALA1

The transition to flowering in Arabidopsis is characterized by the sharp and localized upregulation of APETALA1 (AP1) transcription in the newly formed floral primordia. Both the flower meristem-identity gene LEAFY (LFY) and the photoperiod pathway involving the FLOWERING LOCUS T (FT) and FD genes contribute to this upregulation. These pathways have been proposed to act independently but their respective contributions and mode of interaction have remained elusive. To address these questions, we studied the AP1 regulatory region. Combining in vitro and in vivo approaches, we identified which of the three putative LFY binding sites present in the AP1 promoter is essential for its activation by LFY. Interestingly, we found that this site is also important for the correct photoperiodic-dependent upregulation of AP1. In contrast, a previously proposed putative FD-binding site appears dispensable and unable to bind FD and we found no evidence for FD binding to other sites in the AP1 promoter, suggesting that the FT/FD-dependent activation of AP1 might be indirect. Altogether, our data give new insight into the interaction between the FT and LFY pathways in the upregulation of AP1 transcription under long-day conditions.

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

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

Gene stacking in 1-year-cycling APETALA1 citrus plants for a rapid evaluation of transgenic traits in reproductive tissues

Rapid flowering is crucial to perform functional genomic studies to investigate reproductive biology characteristics and fruit quality-related traits in fruit trees. However, long generation cycles of woody plants considerably delay this evaluation. Through genetic transformation, juvenile periods can be significantly shortened by overexpression of flower meristem-identity genes. Transgenic APETALA1 (AP1) citrus plants behave as rapid-cycling trees, since 1-year-old seedlings promptly show precocious flowering and fruiting. By transgene stacking into these short-generation AP1 and nptII/GUS-positive plants, expression of novel transgenes could theoretically be examined as quickly as 1 year after retransformation. Establishment of the selection and regeneration conditions for the production of retransformed individuals with marker genes is detailed in this communication. Hpt and bar genes were used as the second selectable marker genes. PCR and Southern blot analyses confirmed the recovery of retransformed shoots. AP1 transcript accumulation and GUS and GFP expression were assessed in leaves, and flowers and fruit organs of rapid-cycling retransformed lines, respectively, as early as 1 year after plant generation and during three consecutive years, demonstrating that the principle of stable transgene stacking on early-fruiting transgenic trees is feasible.

An exploration of LEAFY expression in independent evolutionary origins of rosette flowering in Brassicaceae

Whereas most Brassicaceae produce flowers on an elongated inflorescence, a few lineages produce flowers directly from the vegetative rosette on elongated pedicels. Knowing the extent to which independent origins of rosette flowering involve the same developmental and genetic mechanisms could clarify the constraints acting on plant architectural evolution. Prior work in Idahoa, Ionopsidium, and Leavenworthia suggested that changes in the activity or expression of the flower meristem identity gene, LEAFY (LFY), played a role in all three origins of rosette flowering. Here we studied the developmental morphology of L. crassa and immunolocalization of LFY protein in Leavenworthia and Ionopsidium to further compare independent origins of rosette flowering. Leavenworthia crassa differs from Ionopsidium and Idahoa in producing ebracteate flowers. Flowers are, however, associated with "squamules," here interpreted as stipules of a cryptic bract. LFY was detected in L. crassa flower primordia but not in inflorescence meristems. In contrast, the rosette flowering Io. acaule accumulated LFY protein in the inflorescence meristem, whereas its inflorescence-flowering close relative, Io. prolongoi, did not. Thus, although different cases of rosette flowering likely entailed modifications of the same meristem identity program, distinct developmental genetic mechanisms appear to be involved in each case.

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND‐FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene, LEAFY (LFY). Two redundant functioning homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT. Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy, a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.

Ectopic expression of a hyacinth AGL6 homolog caused earlier flowering and homeotic conversion in Arabidopsis

MADS-box genes are involved in floral organ development. Here we report that an AGL6(Agamous-like 6)-like MADS-box gene, HoAGL6, was isolated from Hyacinthus orientalis L. Expression pattern analysis demonstrated that HoAGL6 transcript was detected in inflorescence buds, tepals, carpels and ovules, but not in stamina, leaves or scales. Transgenic Arabidopsis plants ectopically expressing HoAGL6 exhibited novel phenotypes of significantly reduced plant size, extremely early flowering, and losing inflorescence indeterminacy. In addition, wide homeotic conversion of sepals, petals, and leaves into carpel-like or ovary structures, and disappearance or number reduction of stamens in 35S::HoAGL6 Arabidopsis plants were also observed. RT-PCR analysis indicated that the expressions of flowering time gene SOC1 and flower meristem identity gene LFY were significantly up-regulated in 35S::HoAGL6 transgenic Arabidopsis plants, and the expression levels of floral organ identity genes AG and SEP1 in leaves were also elevated. These results indicated that HoAGL6 was involved in the regulation of flower transition and flower organ formation.

Flowering transition in grapevine (Vitis viniferaL.)This review is one of a selection of papers presented at the symposium onVitisat the XVII International Botanical Congress held in Vienna, Austria, 2005.

The available information on the regulation of flowering transition in model systems, such as Arabidopsis and rice, provides a framework to undertake the study of this process in plant species with different growth strategies. The grapevine ( Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world. Understanding the regulation of flowering transition in this species can be relevant for the improvement of yield and quality of the crop. The grapevine is a representative of the family Vitaceae, whose species mostly grow as vines and have evolved climbing organs, tendrils, which are ontogenetically related to the reproductive organs. Here, we summarize the available information on the flowering transition in the grapevine. With this purpose, we first describe the vegetative and reproductive development of the grapevine as well as the reports on the physiology of flowering induction in this species. As well, we review the recent information on the molecular genetics of flowering signal integrator and flower meristem identity genes in the grapevine and compare the process with what is already known in model systems such as Arabidopsis. Finally, we propose a preliminary model to explain the regulation of flower initiation in the grapevine that is useful to identify its differential features and infer future prospects in the understanding of this process.