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Role of auxin and cytokinins in vascular cambium or callus formation

Role of auxin and cytokinins in vascular cambium or callus formation


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Usually, auxins promote cell growth and cytokinins promote cell division. But, I got an information from my teacher that, in case of vascular cambium or callus formation, auxins promote cell division and cytokinins promote cell growth. Is this 'information' true? If true, why?


Role of auxin and cytokinins in vascular cambium or callus formation - Biology

Wed, 11 Dec 2013 04:12:32 +0000

Lipid peroxidation does not always result in the formation of microscopically visible lipofuscin granules, nor is it confined to autophagosomal vesicles it occurs in all functional cell membranes, including the surface membrane. Once the peroxidation of unsaturated lipids is initiated, by haem groups, Fe2+ ions and other simple catalysts in the presence of oxygen, it takes place by a free radical chain reaction. It can be inhibited by lipid-soluble antioxidants such as vitamin E and accelerated by vitamin E deficiency, ionising radiation, chloroform and ethanol poisoning and hyperbaric treatments 8,9, which can cause irreversible damage to cells.

The peroxidation of lipids within cell membranes is occurring in vivo all the time. Some peroxidised lipids may be metabolised but others, perhaps those which are cross linked to other lipids and lipoproteins may not be. The chain reaction of lipid peroxidation may be terminated by the oxidation of other substances which may themselves be damaged and accumulate. Such substances formed within the surface membrane, for example, may accumulate in situ if they are removed from the surface membrane as the membrane is recycled by the invagination of membrane vesicles 11-14 or by other means, some of them might find their way into residual bodies, but they might also be incorporated into intracellular membranes. The formation and accumulation of such substances within the outer and intracellular membranes, for example in the Golgi apparatus, endoplasmic reticulum nuclear membrane and lysosomal membranes, could well be deleterious to normal membrane functioning and could also lead to a positive feedback of damage by further lipid peroxidation, and thus to the senescence and death of the cell. The rate of ageing would be temperature dependent and would also depend on the composition, structure and functions of the cellular membranes, the extra - and intracellular environments, antioxidant levels and so on. Thus, different types of cells would age at different rates but, according to this hypothesis, all cells would be ageing to a greater or lesser extent all the time all cells would be heading towards senescence and death.

The elimination of membranous material from cells might enable the ageing process to be retarded and there are a few examples of the shedding of membranes by cells which I will discuss further. But, in general, the only way in which cells could avoid their otherwise inevitable mortality would be by growing and dividing, thus diluting the accumulated breakdown products. Although lipid peroxidation may be the most important cause of the formation of such substances, the following general considerations could apply to any deleterious substances which accumulate with age.

Growth and division of cells
An artificially simple case is provided by cells dividing symmetrically with a fixed generation time if these accumulate deleterious breakdown products linearly with time, an amount, x, being formed per cell generation time. Successive generations contain more of the accumulated breakdown products but the increments become smaller and smaller. If the rate of accumulation is not linear, but proportional to the amount already accumulated, the content per cell will increase exponentially and if there is a progressive lengthening of the cell generation time, there will be a greater accumulation within individual cells in succeeding generations. With either or both of these assumptions, it can be seen that the whole population will undergo senescence and sooner or later die out.

But another type of cell division is possible, an asymmetrical division in which one of the daughter cells receives all or most of the accumulated breakdown products (becoming more 'mortal') while the other is rejuvenated, receiving little or none. The more 'mortal' of the daughter cells might die or differentiate directly, or it might divide again unequally, producing a rejuvenated cell and a cell even more 'mortal' than itself, or it might undergo one or more sequential symmetrical divisions (as discussed above) to produce a population of cells which sooner or later die (unless they can undergo further asymmetrical divisions to produce rejuvenated cells).

I shall now consider a few aspects of the growth and development of higher plants and higher animals in the light of these ideas. Dicotyledonous trees illustrate the pattern of indefinite growth that is characteristic of plants. (There are of course plants, such as herbaceous annuals, which die after they have flowered. But annuals are capable of growing for much longer than their normal life-span if they are prevented from flowering, indicating that they die because they flower and not because of an innate inability to go on growing 15 .) The life span of trees is limited by a variety of mechanical factors, but cuttings taken from old trees can give rise to healthy young trees, and this process can be repeated indefinitely. The growing points of the tree, the apical meristems, remain perpetually young.

Cell divisions within the apical meristems of the shoots give rise to daughter cells with different fates: some remain meristematic, others give rise to the differentiated structures of the stems and the leaves. Some of these cells die as they differentiate into vascular tissues and fibres, others, for example the leaf mesophyll and pith parenchyma, remain alive for some time, but, unless they are stimulated to divide again in a regenerative response to wounding or damage, they eventually die. The leaves senesce and fall from the tree the pith breaks down. The root meristems give rise to the primary tissues of the root which, apart from those which divide to produce further root meristems, sooner or later die. In secondarily thickening stems the divisions of the cambial cells give rise to cells which die as they differentiate into xylem or undergo further asymmetrical divisions to produce phloem companion cells and sieve tubes. These cells eventually die and are sloughed off in the bark. Cell divisions in the cork cambium give rise to cork cells which die as they differentiate divisions of the root cap initials give rise to root cap cells which die and are sloughed off. Thus, in the various meristems of the plant the continued growth and continued rejuvenation of the meristems is associated with the production of cells which die during or after differentiation.

Vertebrates
Vertebrates, unlike trees, do not go on growing indefinitely, nor can they be propagated vegetatively. At first, fertilised eggs undergo cleavages which rapidly increase the number of cells, but this rate of increase of cell number declines progressively as the animal develops, and as cells and tissues differentiate 16 . Throughout the development of the embryo many tissues and groups of cells regress and die 17,18 . Some of these cell deaths are associated with tissue differentiation 19 , some occur during morphogenetic processes 20 , and others may represent the regression of phylogenetically vestigial structures 17 , but the significance of other cell deaths is obscure. As the animal develops, the cells of some tissues, such as nerve and muscle, differentiate and to a large extent lose the ability to undergo further division. Some of these cells die as the animal grows older and are not replaced 21,22 but in the adult animal a number of other tissues continue to grow, for example the epidermis, the intestinal lining, the liver and blood cells continue to be formed. In all these examples the production of new cells is offset by cell death. Cell divisions in the basal layers of the mammalian epidermis give rise to daughter cells which remain in the basal layers and divide again, and other daughter cells which differentiate and keratinise, dying as they do so. Cell divisions in the crypts of the intestinal villi replenish the population of crypt cells capable of further division and produce other daughter cells which move up the villi where they die and are sloughed off 23 . Asymmetrical divisions of the early precursors of all cells of the blood occur throughout life and give rise to further precursor cells as well as to the maturing and mature cells of the blood, all of which have a limited life span. During the formation of red blood cells 24 and granulocytes 25 in the bone marrow, and lymphocytes in the thymus 26 , considerable numbers of cells die in situ soon after they are formed. The reasons for this 'ineffective' erythropoiesis, granulopoiesis and lymphopoiesis are unknown.

The mortality of at least some of the cells which die in developing animal embryos and in mature animals may represent the price that is paid for the rejuvenation of other cells which continue to grow and divide. But unfortunately too little is known about cell lineages in animals, especially in embryos, for it to be possible to decide how general is the phenomenon of asymmetrical cell divisions giving rise to daughter cells of unequal mortality. The recognition of this pattern is complicated by the fact that by no means all cell death takes place as a result of cellular senescence. Some cells die as they differentiate and others may die because they find themselves in the wrong places at the wrong times 19 . Cell deaths may be controlled chemically, for example by steroid hormones: the injection of glucocorticoids can cause large numbers of lymphocytes to die 27 , the regression of Mullerian and Wolffian ducts is controlled by androgens and oestrogens 19,28 and the regression of the lining of the female genital tract is under the control of oestrogens 28 . But, under the hypothesis that asymmetrical cell divisions lead to a rejuvenation of 'meristematic' daughter cells at the price of the increased mortality of their sister cells, it does not matter whether the latter die as a result of senescence, or whether they die as they differentiate or for any other reason.

Sexual reproduction
In the sexual reproduction of both higher plants and higher animals almost all the cytoplasm from which the embryo and the new organism develops is provided by the egg. In both cases, the egg cells are formed as a result of asymmetrical divisions of the egg mother cell. In the great majority of higher plants, the meiotic divisions of the egg mother cell produce four cells, three of which die. The fourth undergoes further divisions to produce the cells of the embryo sac, most of which die before or shortly after fertilisation. In some species, one of more of the three sister cells of the cell which gives rise to the egg may undergo further division to produce short-lived embryo sac cells29. In animals the first and second meiotic divisions of the egg mother cell give rise to the first and second polar bodies, which regress and die.

It is particularly striking that in both plants and animals, only one of the progeny of the egg mother cell gives rise to an egg while the sister cells die (or if they divide give rise to short-lived progeny). By contrast, there is no comparable cell loss in male gametogenesis associated with the meiotic divisions of the pollen mother cells and spermatogonia.

The many examples in both higher plants and higher animals (and many more can be found in the lower plants and lower animals) of the production of rejuvenated meristematic, stem or egg cells by asymmetrical divisions do not of course prove that these divisions involve an asymmetrical distribution of deleterious breakdown products but the available facts appear to be consistent with this hypothesis.

Loss of membranous material by animal cells
If the accumulation of deleterious breakdown products of membrane lipids is one of the causes of cellular senescence, the loss of membranous material might be of considerable importance in enabling cells to rid themselves of such substances. The shedding of membranous material by living cells does not seem to be of common occurrence but can take place in mammalian cells as follows.

First, in apocrine secretions part of the cell membrane is lost. The best example, and the only one for which conclusive ultrastructural evidence exists, is in the secretion of lipid droplets by the cells of lactating mammary glands. The secreted lipid droplets are surrounded by a unit membrane derived in part from the surface membrane and in part from Golgi vesicle membranes.

Second, membrane-bounded vesicles of cytoplasm can break away from mammalian macrophages both in vitro and in vivo. This process, known as clasmotosis, is of unknown significance. Lymphocytes which are activated in immunological reactions or as a result of phytohaemagglutinin stimulation form 'tails' (uropods) which can bleb off vesiculated buds in vivo and in vitro. Again, the significance of this process is unknown. Clasmotosis is also frequently observed in cultures of fibroblasts.

Third, many types of animal viruses are budded off from host cells in membrane-bounded vesicles. The protein in the membrane of the vesicles is largely viral, at least in the case of RNA tumour viruses, but the lipids are derived from the host cell membrane 35 . Viral particles bounded by membrane are also budded off from the cells of a number of spontaneously cancerous tissues and from many of the cell strains and permanent cells lines which are commonly cultured in laboratories.

Tissue cultures
Many plants callus cultures can be grown indefinitely in vitro. During the early stages of the growth of some calluses, an exponential increase in cell number takes place at a rate which suggests that many of the cells may undergo a limited number of sequential symmetrical divisions before the growth rate declines but in most plant tissue cultures the rate of increase of cell number is more or less linear for most of the growth period 39,40 . Linear growth characteristics would be compatible with a meristematic pattern of cell division such that some daughter cells continue to grow and divide while their sister cells age and sooner or later die. Unfortunately nothing is known in detail about cell lineages within these cultures, nor are there any quantitative data on cell death. Nevertheless, dead and dying cells are by no means uncommon.

'Permanent' mammalian cell lines capable of indefinite propagation in vitro can be derived from cancerous tissues and also from cells which have undergone a spontaneous 'transformation' during culture. Diploid fibroblast cultures can be propagated, however, only for a finite number of subculturings, more (up to about 60) if the cells are derived from embryonic tissues, fewer if they are derived from mature organisms 41 . The number of generations through which the cells can be passed before the population senesces and dies out is reduced if the period of time between the subculturings is increased 42 . Fibroblasts of the mouse L strain have been observed to divide symmetrically over six to seven cell generations with a more or less constant generation time 43 if the cells in the diploid fibroblast cultures also divide symmetrically, deleterious breakdown products might accumulate in the cells of succeeding generations, as discussed above, and account for the senescence of these cultures. It is impossible, however, to make any detailed interpretation of the senescence of these cultures in the absence of quantitative information about the proportions of dividing and nondividing cells, the incidence of cell death, and the extent and significance of clasmotosis within these cultures - or indeed with cultures of 'transformed' and 'permanent' cell lines.

Cancer
Malignancy must not only involve the freeing of cells from the normal controls on their proliferation, but also the avoidance of senescence by at least a part of the cell population. Many animal tumours contain a stem cell or 'meristematic' population which gives rise to daughter cells which may or may not differentiate, but which sooner or later die. There are numberous examples of cell death within cancerous tissues 45-48 . Some of the cell deaths can be explained in terms of an inadequate vascularisation of the tumour tissue, but in most tumours this is by no means the only cause an does not apply to all to leukaemias many of the cells may die as a result of ageing.

Little attention has been paid to the incidence of cell death within cultures of cancerous cells and it is therefore at present impossible to know to what extent the patterns of cell division, ageing and death within these cultures resemble those within in vivo cancers. It is sometimes assumed, if only implicitly, that overall exponential growth characteristics of cell cultures mean that there is a homogeneous population of symmetrically dividing cells. This assumption is not justified: a heterogeneous population containing proliferating, nonproliferating and dying cells can also grow exponentially if the proportion of cells that die is constant with time.

It is conceivable that the loss of membranous material either spontaneously, as in certain types of mammary gland tumours, or as a result of the budding off of viruses (such as RNA tumour viruses) could play a significant role in the retardation of cellular senescence in certain types of cancer.

Effects of cell death
Very little is known about the biochemistry of dying cells. Such cells probably release all sorts of proteins, glycoproteins, peptides, amino acids, amino acid breakdown products, nucleic acids and nucleic acid breakdown products, lipids and lipid breakdown products as well as salts and other substances which were sequestered inside the cells.

It has recently been found that in higher plants the hormone auxin (indole-3-acetic acid) is formed as a consequence of cell death as tryptophan, released by proteolysis, is broken down. Dying cells in differentiating vascular tissue, regressing nutritive tissues and so on, are probably the major source of this hormone within the plant 52 . Other plant hormones may also be produced by damaged and dying cells: ethylene from the breakdown of methionine and cytokinins by the hydrolysis of transfer RNA. In higher plants the normal production of hormones as a consequence of cell death and the production of 'wound hormones' by damaged cells can be seen as two aspects of the same phenomenon. 52

Wound and regenerative responses in vertebrates cannot be explained simply in terms of wound hormones, but there is evidence that dying cells release substances that stimulate phagocytosis 53 , and affect growth and development in both normal 54,55 and cancerous tissues 56 . And at least some of the cell deaths which occur during normal embryonic development may well result in the production or release of substances involved in the control of differentiation and development.

Dying cells may not only have a chemical effect on neighbouring cells but also a physical effect as cell to cell contacts are broken. Cell deaths within a tissue may also affect the functioning of the tissue as a whole: for example, the death of nerve cells within the brain 22 seems likely to affect pathways or patterns of nervous conduction, perhaps leading to the formation of new pathways or patterns. Such cell deaths could act as a source of random change within the nervous system that might not always be deleterious 57 .

So little attention has been paid to the ageing and death of cells during growth and development, both normal and abnormal, that detailed information about these processes is scarce. Where facts are few, speculation can flourish. Most of the speculations advanced in this article could be opposed by alternative speculations, but they illustrate the view that growth and development cannot be understood in isolation from ageing and death. This is by no means an original concept, but at the cellular level it provides a perspective in which many familiar facts take on a new significance and suggests a new approach to familiar problems.


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Introduction

Vascular tissues in plants are either primary or secondary. Primary xylem and phloem differentiate within the vascular cylinder during primary growth of stems and roots. Later in development, when elongation ceases and plants undergo secondary growth, a vascular cambium develops that gives rise to secondary xylem and secondary phloem ( Esau, 1965 ). The dividing cambial initials produce phloem or xylem mother cells which subsequently undergo one or several rounds of division before differentiation. The ‘cambial zone’ includes the cambial initials and phloem and xylem mother cells ( Lachaud et al., 1999 Larson, 1994 ). During both primary and secondary growth, positional information is necessary to coordinate the spatio-temporal formation of vascular strands not only throughout the plant organ but also within vascular bundles. Our current knowledge concerning the molecular mechanisms underlying many aspects of vascular development, such as control of cambial cell division, primary and secondary xylem/phloem differentiation and patterning, is still fragmented.

It is well known that plant hormones, and in particular auxin, play a crucial role in the developmental control of primary and secondary vascular tissues and cambial activity ( Little and Pharis, 1995 Ye, 2002 ) by regulating both radial ( Sundberg et al., 2001 Uggla et al., 1996 ) and longitudinal vascular pattern formation in plants ( Berleth et al., 2000 Sieburth, 1999 ). Developing buds and young shoots are major sources of auxin, but indole acetic acid (IAA) is also synthesized in young root tissues ( Ljung et al., 2002 ). Auxin is transported through the plant in a basipetal polar fashion (for reviews see Lomax et al., 1995 Swarup and Bennett, 2003 ). In the canalization hypothesis proposed by Sachs (1981) this polar auxin transport has been related to the differentiation of provascular strands. In recent years, genetic studies of Arabidopsis mutants altered in auxin metabolism have increased our understanding of auxin transport and signal transduction in relation to vascular differentiation and patterning (for reviews see Berleth and Mattsson, 2000 Berleth et al., 2000 Hobbie, 1998 ). It is known that polar auxin transport is mediated to a great extent by the efflux carrier PIN1. Based on the pin1 mutant phenotype, it was also demonstrated that auxin transport has a central role in the formation of vascular strands ( Gälweiler et al., 1998 ). Many proteins involved in responses to auxin have been characterized, and at least two of them, PINOID, a putative protein kinase, and MP, an auxin response factor, have been shown to be important for vascular differentiation and patterning ( Christensen et al., 2000 Hardtke and Berleth, 1998 ).

Although there is some evidence pointing to a role for cytokinin in procambial cell division and xylem differentiation, its mode of action is not well understood ( Saks et al., 1984 for review see Aloni, 1995 ). In vitro studies of xylogenesis in Zinnia elegans indicate that auxin alone is not sufficient to induce the differentiation of leaf mesophyll cells into tracheary elements (TE), and that cytokinin is a strict requirement for differentiation of TE ( Fukuda and Komamine, 1980 ). More recently, the characterization of the wooden leg (wol) mutant and the subsequent cloning of the corresponding gene, CYTOKININ RESPONSE (CRE1), demonstrated that cytokinin regulates vascular development by controlling procambial cell division ( Inoue et al., 2001 Mähönen et al., 2000 ). As a result of defective procambial cell divisions, all procambial cells in roots differentiate uniquely into protoxylem, with neither protophloem nor metaxylem forming in the stele. The WOL/CRE1 gene encodes a cytokinin receptor which belongs to the two-component signal transducer family. It is expressed in procambial cells of primary roots.

Although we are still a long way from establishing an integrated model that thoroughly explains the differentiation of cambial cells into functional vascular bundles, important headway has been made in dissecting certain steps of vascular differentiation and patterning per se by carrying out mutant screening for vascular defects. Many mutants with a variety of phenotypes have been characterized (for reviews see Fukuda, 2004 Scarpella and Meijer, 2004 Turner and Sieburth, 2002 ). In some case, mutant analysis has resulted in a more precise function for some genes already known to be involved in secondary cell deposition. This is the case for irx3 encoding cellulose synthase ( Taylor et al., 1999 ) and irx4 encoding cinnamoyl Co-A reductase ( Jones et al., 2001 ). In others cases, genetic analysis has revealed the role of important regulatory genes in vascular formation. Recently, a MYB transcription factor, ALTERED PHLOEM DEVELOPMENT (APL) has been identified as a key player in phloem development ( Bonke et al., 2003 ). Another regulatory gene INTERFASCICULAR FIBERLESS1 (IFL1), belonging to the homeodomain-leucine zipper protein (HD-ZIP) is essential for proper fiber differentiation in Arabidopsis stems ( Ratcliffe et al., 2000 Zhong and Ye, 1999 Zhong et al., 1997 ). Although numerous vascular mutants have been described, to our knowledge mutants with altered cambial activity and/or secondary growth are extremely rare ( Oyama et al., 1997 ).

Contrary to preconceived notions, Arabidopsis is considered to be an excellent model for the study of secondary growth ( Chaffey et al., 2002 ). Under normal growth conditions, hypocotyls of mature plants possess a vascular cambium and undergo extensive secondary growth ( Busse and Evert, 1999 Ye et al., 2002 ). Secondary growth has also been described in the inflorescence stem ( Altamura et al., 2001 ), and this phenomenon is enhanced when plants are grown at a low population density and/or with repeated removal of all newly emerging inflorescences stems. Taking advantage of this characteristic, Zhao et al. (2000) constructed a cDNA library from root-hypocotyl sections enriched in developing xylem cells. Moreover, genes related to secondary growth were identified by hybridizing the Arabidopsis Genome GeneChip arrays with cDNA extracted from stems with enhanced secondary xylem formation ( Ko et al., 2004 Oh et al., 2003 ).

Since Arabidopsis possesses a vascular cambium and undergoes secondary growth it should be possible to identify molecular and physiological factors that control cambial activity by identifying mutants altered in amounts or patterns of secondary vascular tissues. An assumption may be made that these alterations would be the direct consequence of altered cambial activity. To address this issue, we have screened a T-DNA collection of Arabidopsis and isolated a mutant that has abnormally high secondary xylem production in stems. In hca (for ‘high cambial activity’), the extensive secondary growth altered the organization of the stem vasculature leading to a continuous ring of vascular tissues the mutant is impaired in cambial activity and secondary growth throughout the plant body. We have demonstrated by multiple, independent assays that responses to both auxin and cytokinin were affected in the hca mutant.


3. The Role of WOX Genes during Somatic Embryogenesis and De Novo Organogenesis in Conifers

WOX genes constitute a plant-specific homeobox family whose members have important functions during plant growth and development, such as embryo patterning, organ formation and stem cell maintenance. Phylogenetic analyses carried out by van der Graaff et al. [67] have established three distinct clades in the WOX gene family: the ancient clade, whose members are present in all plant lineages from green algae to seed plants the intermediate clade, present in vascular plants and the modern or WUS clade, only found in ferns and seed plants. The WOX gene family includes 14 members in Pinus pinaster and 13 in Picea abies distributed throughout the three clades previously mentioned [68,69]. The analysis of their expression during SE and in different plantlet tissues by quantitative real-time PCR (RT-qPCR), RNA sequencing and in situ mRNA hybridization showed that the expression profiles of WOX genes in conifers are quite similar to those described for their angiosperm counterparts ( Figure 3 ), suggesting a high degree of conservation of the gene family across seed plants [68,69]. WOX gene family diversity in Arabidopsis thaliana and several gymnosperm species are presented in more detail in Table 1 at the end of this section.

Table 1

List of genes belonging to the WUSCHEL-RELATED HOMEOBOX (WOX) family, including those from model species Arabidopsis thaliana and their homologue genes already identified in gymnosperms, with name abbreviation, locus code (AGI code in case of Arabidopsis thaliana, GenBank number in case of gymnosperm species), function, location and references. Shoot apical meristem, SAM root apical meristem, RAM.

SpeciesName AbbreviationLocus CodeFunction and LocationReferences
i. WUS clade
Arabidopsis thaliana AtWOX1AT3G18010Lateral organ primordia formation[75,84,85]
AtWOX2AT5G59340Apical embryo and embryo patterning[75,76]
AtWOX3/PRSAT2G28610SAM, lateral organ formation[81]
AtWOX4AT1G46480Vascular tissue, procambial development[82]
AtWOX5AT3G11260Stem cell maintenance (RAM)[80]
AtWOX6AT2G01500Cold-stress response[83]
AtWOX7AT5G05770Lateral root development[86]
AtWUSAT2G17950Stem cell maintenance (SAM)[79]
Ginkgo biloba GbWOX2 <"type":"entrez-nucleotide","attrs":<"text":"FM882124","term_id":"229359434","term_text":"FM882124">> FM882124Embryo patterning[88]
GbWOX3A <"type":"entrez-nucleotide","attrs":<"text":"FM882125","term_id":"229359436","term_text":"FM882125">> FM882125Lateral organ outgrowth[88]
GbWOX3B <"type":"entrez-nucleotide","attrs":<"text":"FM882126","term_id":"229359290","term_text":"FM882126">> FM882126Lateral organ outgrowth[88]
GbWOX4 <"type":"entrez-nucleotide","attrs":<"text":"HF564615","term_id":"531033846","term_text":"HF564615">> HF564615Germinating embryo, vascular cambium[88]
GbWUS <"type":"entrez-nucleotide","attrs":<"text":"FM882128","term_id":"229359294","term_text":"FM882128">> FM882128Embryo, shoot tip[88,90]
Gnetum gnemon GgWOX2A <"type":"entrez-nucleotide","attrs":<"text":"HF564611","term_id":"531033838","term_text":"HF564611">> HF564611Embryo patterning[88]
GgWOX2B <"type":"entrez-nucleotide","attrs":<"text":"HF564619","term_id":"531033854","term_text":"HF564619">> HF564619Embryo patterning[88]
GgWOX4 <"type":"entrez-nucleotide","attrs":<"text":"HF564612","term_id":"531033840","term_text":"HF564612">> HF564612Germinating embryo, vascular cambium[88]
GgWOX6/WOXX <"type":"entrez-nucleotide","attrs":<"text":"HF564620","term_id":"531033856","term_text":"HF564620">> HF564620n/a[88]
GgWOXY <"type":"entrez-nucleotide","attrs":<"text":"HF564621","term_id":"531033858","term_text":"HF564621">> HF564621n/a[88]
GgWUS <"type":"entrez-nucleotide","attrs":<"text":"FM882154","term_id":"229359346","term_text":"FM882154">> FM882154Embryo, shoot tip[88,90]
Picea abies PaWOX2 <"type":"entrez-nucleotide","attrs":<"text":"AM286747","term_id":"110321611","term_text":"AM286747">> AM286747Embryo patterning[68,71,72,73]
PaWOX3 <"type":"entrez-nucleotide","attrs":<"text":"JX411947","term_id":"498904042","term_text":"JX411947">> JX411947Lateral organ outgrowth[68,89]
PaWOX4 <"type":"entrez-nucleotide","attrs":<"text":"JX411948","term_id":"498904044","term_text":"JX411948">> JX411948Germinating embryo, vascular cambium[68]
PaWOX5 <"type":"entrez-nucleotide","attrs":<"text":"JX411949","term_id":"498904046","term_text":"JX411949">> JX411949Embryo, SAM, RAM[68]
PaWOXX <"type":"entrez-nucleotide","attrs":<"text":"KX011459","term_id":"1026299348","term_text":"KX011459">> KX011459Embryo, SAM, needles[69]
PaWUS <"type":"entrez-nucleotide","attrs":<"text":"JX512364","term_id":"498905387","term_text":"JX512364">> JX512364Embryo, shoot tip[68]
Pinus pinaster PpWOX2 <"type":"entrez-nucleotide","attrs":<"text":"KU962991","term_id":"1026299306","term_text":"KU962991">> KU962991Embryo patterning[69]
PpWOX3 <"type":"entrez-nucleotide","attrs":<"text":"KU962992","term_id":"1026299308","term_text":"KU962992">> KU962992Lateral organ outgrowth[69]
PpWOX4 <"type":"entrez-nucleotide","attrs":<"text":"KU962993","term_id":"1026299310","term_text":"KU962993">> KU962993Germinating embryo, vascular cambium[69]
PpWOX5 <"type":"entrez-nucleotide","attrs":<"text":"KT356216","term_id":"950805367","term_text":"KT356216">> KT356216Embryo, SAM, RAM[69]
PpWOXX <"type":"entrez-nucleotide","attrs":<"text":"KU962995","term_id":"1026299314","term_text":"KU962995">> KU962995Embryo, SAM, needles[69]
PpWUS <"type":"entrez-nucleotide","attrs":<"text":"KT356213","term_id":"1003330093","term_text":"KT356213">> KT356213Embryo, shoot tip[69]
Pinus sylvestris PsWOX2 <"type":"entrez-nucleotide","attrs":<"text":"FM882159","term_id":"229359432","term_text":"FM882159">> FM882159Embryo patterning[90]
PsWOX3 <"type":"entrez-nucleotide","attrs":<"text":"FM882158","term_id":"229359354","term_text":"FM882158">> FM882158Lateral organ outgrowth[90]
PsWOX4 <"type":"entrez-nucleotide","attrs":<"text":"HF564616","term_id":"531033848","term_text":"HF564616">> HF564616Germinating embryo, vascular cambium[90]
PsWOX5/WUS <"type":"entrez-nucleotide","attrs":<"text":"FM882160","term_id":"229359356","term_text":"FM882160">> FM882160Embryo, SAM, RAM[90]
Pinus taeda PtWOX2 <"type":"entrez-nucleotide","attrs":<"text":"KX011449","term_id":"1026299328","term_text":"KX011449">> KX011449Embryo patterning[69]
PtWOX3 <"type":"entrez-nucleotide","attrs":<"text":"KX011450","term_id":"1026299330","term_text":"KX011450">> KX011450Lateral organ outgrowth[69]
PtWOX4 <"type":"entrez-nucleotide","attrs":<"text":"KX011451","term_id":"1026299332","term_text":"KX011451">> KX011451Germinating embryo, vascular cambium[69]
PtWOX5 <"type":"entrez-nucleotide","attrs":<"text":"KX011452","term_id":"1026299334","term_text":"KX011452">> KX011452Embryo, SAM, RAM[69]
PtWOXX <"type":"entrez-nucleotide","attrs":<"text":"KX011454","term_id":"1026299338","term_text":"KX011454">> KX011454Embryo, SAM, needles[69]
PtWUS <"type":"entrez-nucleotide","attrs":<"text":"KX011458","term_id":"1026299346","term_text":"KX011458">> KX011458Embryo, shoot tip[69]
ii. Intermediate clade
Arabidopsis thaliana AtWOX8/STPLAT5G45980Basal embryo patterning[75,76]
AtWOX9/STIMPYAT2G33880Basal embryo patterning, cell proliferation[75]
AtWOX11AT3G03660Adventitious root formation[78]
AtWOX12AT5G17810De novo root organogenesis[78]
Ginkgo biloba GbWOX9 <"type":"entrez-nucleotide","attrs":<"text":"HF564618","term_id":"531033852","term_text":"HF564618">> HF564618n/a[88]
Gnetum gnemon GgWOX9 <"type":"entrez-nucleotide","attrs":<"text":"HF564613","term_id":"531033842","term_text":"HF564613">> HF564613n/a[88]
Picea abies PaWOX8/9 <"type":"entrez-nucleotide","attrs":<"text":"GU944670","term_id":"294818269","term_text":"GU944670">> GU944670Embryo patterning[68,73,77]
PaWOX8A <"type":"entrez-nucleotide","attrs":<"text":"JX411950","term_id":"498904048","term_text":"JX411950">> JX411950Embryo patterning[68]
PaWOX8B <"type":"entrez-nucleotide","attrs":<"text":"JX411951","term_id":"498904050","term_text":"JX411951">> JX411951Embryo patterning[68]
PaWOX8C <"type":"entrez-nucleotide","attrs":<"text":"JX411952","term_id":"498904052","term_text":"JX411952">> JX411952Embryo patterning[68]
PaWOX8D <"type":"entrez-nucleotide","attrs":<"text":"JX411953","term_id":"498904054","term_text":"JX411953">> JX411953Embryo patterning[68]
Pinus pinaster PpWOXB <"type":"entrez-nucleotide","attrs":<"text":"KU962997","term_id":"1026299318","term_text":"KU962997">> KU962997Embryo patterning[69]
PpWOXC <"type":"entrez-nucleotide","attrs":<"text":"KU962998","term_id":"1026299320","term_text":"KU962998">> KU962998Embryo patterning[69]
PpWOXD <"type":"entrez-nucleotide","attrs":<"text":"KU962999","term_id":"1026299322","term_text":"KU962999">> KU962999Embryo patterning[69]
PpWOXE <"type":"entrez-nucleotide","attrs":<"text":"KU963000","term_id":"1026299324","term_text":"KU963000">> KU963000Embryo patterning[69]
PpWOXF <"type":"entrez-nucleotide","attrs":<"text":"KU963001","term_id":"1026299326","term_text":"KU963001">> KU963001Embryo[69]
Pinus sylvestris PsWOX9 <"type":"entrez-nucleotide","attrs":<"text":"FM882155","term_id":"229359348","term_text":"FM882155">> FM882155n/a[90]
Pinus taeda PtWOXB <"type":"entrez-nucleotide","attrs":<"text":"KX011456","term_id":"1026299342","term_text":"KX011456">> KX011456Embryo patterning[69]
PtWOXE <"type":"entrez-nucleotide","attrs":<"text":"KX011457","term_id":"1026299344","term_text":"KX011457">> KX011457Embryo patterning[69]
iii. Ancient clade
Arabidopsis thaliana AtWOX10AT1G20710n/a[67,70]
AtWOX13AT4G35550Floral transition, root development[70]
AtWOX14AT1G20700Floral transition, root development[70]
Ginkgo biloba GbWOX13 <"type":"entrez-nucleotide","attrs":<"text":"HF564617","term_id":"531033850","term_text":"HF564617">> HF564617n/a[88]
Gnetum gnemon GgWOX13 <"type":"entrez-nucleotide","attrs":<"text":"HF564614","term_id":"531033844","term_text":"HF564614">> HF564614n/a[88]
Picea abies PaWOX13n/an/a[68]
PaWOXG <"type":"entrez-nucleotide","attrs":<"text":"MG545153","term_id":"1318843738","term_text":"MG545153">> MG545153n/a[69]
Pinus pinaster PpWOX13 <"type":"entrez-nucleotide","attrs":<"text":"KU962994","term_id":"1026299312","term_text":"KU962994">> KU962994n/a[69]
PpWOXA <"type":"entrez-nucleotide","attrs":<"text":"KU962996","term_id":"1026299316","term_text":"KU962996">> KU962996n/a[69]
PpWOXG <"type":"entrez-nucleotide","attrs":<"text":"MG545154","term_id":"1318843740","term_text":"MG545154">> MG545154n/a[69]
Pinus sylvestris PsWOX13 <"type":"entrez-nucleotide","attrs":<"text":"FM882156","term_id":"229359350","term_text":"FM882156">> FM882156n/a[90]
Pinus taeda PtWOX13 <"type":"entrez-nucleotide","attrs":<"text":"KX011453","term_id":"1026299336","term_text":"KX011453">> KX011453n/a[69]
PtWOXA <"type":"entrez-nucleotide","attrs":<"text":"KX011455","term_id":"1026299340","term_text":"KX011455">> KX011455n/a[69]
PtWOXG <"type":"entrez-nucleotide","attrs":<"text":"MG545155","term_id":"1318843742","term_text":"MG545155">> MG545155n/a[69]

n/a: non available information.

Schematic representation of the expression domains of some WOX and KNOX genes in conifers according to quantitative real-time PCR, RNA sequencing RNA-seq and in situ mRNA hybridization results. (A) Shoot apex (B) late and mature somatic embryo. Source: unpublished drawings from the authors.

Ancient-clade genes are constitutively expressed in all developmental stages of SE but also in all plantlet tissues analyzed in Picea abies and Pinus pinaster [68,69] (see Supplementary Figure S1), which is consistent to what was previously reported in angiosperms [70], although their function in conifers still remains unknown. In contrast, the WUS-clade member WOX2 and most members from the intermediate clade are mainly expressed during early and late SE, with low expression levels in mature somatic embryos, both in Picea abies and Pinus pinaster [68,69]. Besides, expression of PaWOX2 was also detected by in situ mRNA hybridization in immature zygotic embryos in Picea abies, but not in the mature ones [71]. However, practically no expression was found during zygotic embryo germination or in plantlets for WOX2 and most intermediate members in the analyzed coniferous species. Based on this expression pattern, WOX2 has been proposed as a good marker of early stages of SE in Picea abies [72,73]. For example, WOX2 allowed distinguishing EMs from non-embryogenic calli during SE from primordial shoots in Picea glauca [32]. Similarly, this gene was only expressed in EMs derived from shoots buds and immature zygotic embryos, but not in non-embryogenic callus induced from young needles of 1-month-old seedlings in Pinus contorta [74].

Orthologues of these genes in Arabidopsis thaliana, AtWOX2 and the members from the intermediate clade AtWOX8 and AtWOX9 are involved in early embryonic pattern formation [75,76]. Basically, AtWOX2 and AtWOX8 are expressed in the female gametophyte and zygote. After the first division AtWOX2 transcripts are only detected in the apical daughter cell that will originate the embryo proper, while AtWOX8 expression is restricted to the basal daughter cell that will give rise to the embryo suspensor and the hypophyseal cell, establishing in that way the apical-basal polarity of the embryo. For its part, AtWOX9 also contributes to the embryo polarity, as it is expressed initially in the hypophysis and then expands into the central domain of the embryo. In Picea abies, PaWOX2 and the intermediate-clade member PaWOX8/9 have been also shown to participate in the establishment of the apical-basal embryo pattern during early embryo development [71,77]. In order to unravel their role in this process, RNA interference (RNAi) lines for each gene were constructed using both constitutive and inducible promoters. Downregulation of PaWOX2 and PaWOX8/9 through RNAi during the first stages of SE results in aberrant embryos due to the lack of a well-defined border between the globular EM and the suspensor, failing to form mature somatic embryos at a higher frequency than the control lines. In both cases, the effects of inhibiting their expression are observed mainly during early embryo differentiation, and practically no defects were observed when downregulation takes place after late embryo formation. In the case of PaWOX8/9, an alteration of the cell division planes in the basal cells of the EM, and the differentiation of suspensor cells (both basal and top cells), was observed by confocal microscopy [77]. In fact, it was reported that PaWOX8/9 RNAi lines showed altered expression levels of several cell-cycle-regulating genes. Whereas PaWOX8/9 regulates cell division at the transcriptional level and cell fate determination, downregulation of PaWOX2 does not affect the expression of the genes that participate in the regulation of the cell cycle [71]. Instead of that, high expression levels of PaWOX2 are required during early embryogenesis for the correct development of the protoderm, the external layer of the globular embryo which will give rise to the epidermis, in early and late embryos. Furthermore, this gene has been shown to be essential for the expansion of the suspensor cells during early embryo development. Other members from the intermediate clade in conifers are phylogenetically close to AtWOX11 and AtWOX12, which have been related to root organogenesis [78], although no information about their role in conifers is still available.

The WUS clade in conifers contains orthologues of the genes WUS, WOX5, WOX3 and WOX4 previously described in angiosperms [68,69]. In Arabidopsis thaliana, these genes have been involved in the maintenance of stem cells in the SAM, root apical meristem (RAM), leaf marginal meristems and procambium, respectively [79,80,81,82] (see Supplementary Figure S1). However, no orthologues have been found for AtWOX1, AtWOX6 and AtWOX7, which have been shown to participate in lateral organ primordia formation, cold-stress responses and lateral root development, respectively [83,84,85,86].

In conifers, WUS expression is low during the first stages of SE and reaches a peak in somatic mature embryos, when the SAM is already established [68,69]. In 3-week-old plantlets, transcripts were detected exclusively in a small group of cells situated in the central zone of the SAM through RT-qPCR and in situ mRNA hybridization [69], which might indicate that PpWUS regulates the balance between proliferation and differentiation of stem cells, similarly to what was established in angiosperms. Interestingly, the effects of inducible ectopic expression of AtWUS were analyzed in different stages of SE, germinating somatic embryos and seedlings in Picea glauca [87]. Expression of AtWUS caused important alterations during somatic embryo formation. In germinating embryos, induction of AtWUS expression inhibited root growth, but normal shoot development was observed, supporting the participation of this gene in SAM maintenance. In contrast to Arabidopsis thaliana, expression of AtWUS did not induce ectopic shoot formation on Picea glauca seedlings. It is noticeable that the WUS clade in gymnosperms contains a gene absent in angiosperms called WOXX, whose expression profile during SE and in plantlets in Pinus pinaster is similar to that described for PpWUS [69,88].

On the other side, analyses of conifer WOX3 orthologues suggest their involvement in lateral organ formation and differentiation, but not in meristem formation. Expression of PaWOX3 was very low during early and late embryogenesis in Picea abies, reaching its highest value in mature somatic embryos [89]. In particular, these authors detected PaWOX3 expression at the base and lateral margins of cotyledons from mature embryos through in situ mRNA hybridization and GUS staining in pPaWOX3:GUS lines. Furthermore, downregulation of PaWOX3 through RNAi did not affect somatic embryo formation, but alters their cotyledon morphology. In three-week-old plantlets of Pinus pinaster, Alvarez et al. [69] detected PpWOX3 transcripts in lateral organs and in the peripheral zone of the SAM, where organ initiation takes place (see Figure 3 B and Supplementary Figure S1).

Before WUS functionality in the SAM was established, some authors proposed that WOX5 regulated stem cell maintenance both in the SAM and RAM in conifers [68,90]. This hypothesis was based on the fact that WOX5 transcripts were detected by RT-qPCR mainly in root apexes but also in shoot apexes in several coniferous species, whereas no WUS expression was detected in any tissues or developmental stages at that moment. However, as we mentioned before, recent studies have determined that WUS and WOX5 exert their functions of stem cell regulators in the SAM and RAM, respectively, in conifers [69]. Although current evidence support that the functional differentiation of WUS and WOX5 took place before the gymnosperm𠄺ngiosperm split, it cannot be discarded an additional role of WOX5 in conifer SAM functioning based on its expression pattern during SE and in plantlets (see Supplementary Figure S1). Similar to WUS, WOX5 also reaches maximum expression levels during SE in mature embryos in Picea abies and Pinus pinaster, and expression of this gene was also detected in shoot apexes of plantlets [68,69]. In addition, recent interspecies complementation experiments have shown that the expression of both WUS and WOX5 orthologues from different gymnosperm species under the control of AtWUS and AtWOX5 promoters can rescue the phenotypes of the Arabidopsis wus-1 and wox5-1 loss-of-function mutants [91]. These findings suggest that gymnosperm WUS and WOX5 proteins are interchangeable when expressed under the right conditions, as it had been previously established in angiosperms [92].

Based on these results, Alvarez et al. [93] analyzed the expression pattern of PpWUS, PpWOXX and PpWOX5 during the induction phase of in vitro caulogenesis in Pinus pinea to determine their participation in de novo shoot meristem formation. In particular, transcript levels of these genes, among others, were measured in Pinus pinea cotyledons cultured on the presence and absence of 44.4 µM BA during short and long times of culture (0𠄱 d and 2𠄶 d, respectively) and analyzed by principal component analysis. The authors found that no PpWOXX expression was detected along the process, whereas PpWUS seems to have an important role at long times of induction. In Arabidopsis thaliana, it was also reported that cytokinin signaling eventually lead to the upregulation of WUS during the induction phase of de novo shoot organogenesis in the center of the incipient shoot meristem [94,95,96]. Expression data were also analyzed in Pinus pinea cotyledons together with the endogenous content of several PGRs by partial least squares regression. Results reinforced the participation of PpWUS in the organogenic induction at long times of culture, but also pointed out that PpWOX5 has a relevant participation in this process, although its exact role still remains unknown.


DOES AUXIN TRANSPORT CONTROL STEM VASCULAR DEVELOPMENT AND PATTERNING?

Examining the role of auxin transport in the vascular patterning of intact plants using mutants disrupted in auxin transport or hormone application studies is complicated by the fact that auxin influences many important developmental processes. This means that it is difficult to determine if the changes in vascular patterning observed are directly related to auxin transport or are due to secondary effects. This could include changes in plant development (e.g. cell size, division and organ shape) that then indirectly influence vascular development, or changes in the level of or response to other hormones influenced by auxin (e.g. gibberellin Reid and Ross, 2011). Indeed, quite different outcomes for vascular development and patterning in the stem can be induced by mutations that disrupt auxin transport in intact plants. For example, altered xylem differentiation and a reduction in the number of vascular bundles occur in the inflorescence stem of the arabidopsis aux1 lax1 lax2 lax3 quadruple auxin influx mutant compared with comparable wild-type plants ( Frabregus et al., 2015). In contrast, the auxin efflux mutant pin1, which has a reduction in auxin sources (i.e. young leaves) and auxin transport in stem segments, has a vascular phenotype similar to that of plants that overproduce auxin, with increased xylem production and vascular bundle development ( Galweiler et al., 1998 Benjamins and Scheres, 2008). This is also seen in pin1 pin2 double mutants ( Fabregas et al., 2015). In intact plants, auxin transport inhibitors such as 1-naphthylphthalamic acid (NPA) have also been shown to not only impair vascular continuity but also increase the amount of vascular tissue compared with plants grown on a non-NPA medium ( Mattsson et al., 1999).

Perhaps some of the strongest evidence for auxin transport controlling post-embryonic stem vascular development has been obtained through studies using wounded or grafted plants. Plants have evolved wound response mechanisms that enable survival after herbivory, mechanical damage or pathogen attack ( Asahina et al., 2011 Reid and Ross, 2011 Ikeuchi et al., 2013). The amazing ability of plants to restore vascular connections has also been harnessed in horticultural grafting practices to produce superior plants made up of selected root and shoot combinations ( Mudge et al., 2009 Melnyk and Meyerowitz, 2015). Wounding and grafting studies sever existing vascular strands within the plant stem and allow the observation of the reconnection of the vasculature, either around the wound, through the wound site or across the graft junction ( Sauer et al., 2006 Asahina et al., 2011 Sawchuck and Scarpella, 2013 Fig. 2). In incomplete severing of the stem, new vascular tissue will often form around the wound sites through pre-existing parenchyma tissue or through callus ( Fig. 2A). In the case of complete severing of the vascular tissue, as occurs in grafting, new vascular tissue must form across a callus layer, a mass of totipotent parenchyma cells that aids in binding the plant sections together ( Fig. 2A).

Sachs’ pioneering experiments have shown that in the presence of applied auxin, cells appear to be capable of xylem specification and differentiation ( Sachs, 1969 Fig. 1). Auxin application to grafts may accelerate successful graft unions ( Shimomura and Fujihara, 1977). Melnyk et al. (2015) have proposed that auxin and/or sucrose may drive reconnection and the wound response in the rootstock. The auxin canalization model of vascular development would also predict that disruption of PAT by chemical inhibitors would result in altered vascular patterning. While the PAT inhibitor NPA disrupts key PAT efflux proteins, grafts with NPA applied to the graft junction showed no disruption to phloem reconnection or reduced vascular strand reconnection across the graft junction ( Melynk et al., 2015). Although NPA may not entirely block auxin transport ( Bishopp et al., 2011), this result suggests that reducing PAT may not necessarily reduce vascular reconnection. However, use of another auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA), did suppress tissue reunion in incised inflorescence stems ( Asahina et al., 2011) and reduced the width and length of vascular tissue formed at the graft junction in arabidopsis ( Matsuoka et al., 2016).


Acknowledgements

We thank Ykä Helariutta (University of Helsink, Finland), Hiroo Fukuda (The University of Tokyo, Japan) and Jing-Chu Luo (Peking University, China) for the valuable suggestions on this project and critical comments on the manuscript. We thank Sedeer El-Showk (University of Helsink, Finland) for critical reading and editing of the manuscript. This work was supported by the National Natural Science Foundation of China (31070156), the China Ministry of Agriculture Transgenic Breeding Projects (no. 2009ZX08009-095B) and the China Ministry of Science and Technology 863 Program (no. 2006AA02Z334 2007AA02Z165).

Fig. S1 The regenerated sieve elements at stage II during secondary vascular tissue regeneration.

Fig. S2 Enriched GO categories during SVT regeneration.

Table S1 Expression data of genes involved in epigenetic regulation and cell cycle

Table S2 Expression data of genes involved in xylem development

Table S3 Expression data of genes involved in phloem and cambium specification

Table S4 Expression data of genes involved in phytohormones

Table S5 Primers for qRT-PCR

Methods S1 Additional information for experimental procedure.

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Acknowledgements

The authors thank David Baulcombe (Cambridge University) and Yi Li (Peking University) for providing the TRV vectors for the VIGS experiments, Jinfang Chu (Institute of Genetics and Developmental Biology, CAS) for the IAA measurements, Lijia Qu and John Olson (Peking University) for proofreading the manuscript. This work was supported by the National Key Basic Research Program of China (2012CB114500) and the National Natural Science Foundation of China (31270219).


Reconstitutive approach for investigating plant vascular development

Plants generate various tissues and organs via a strictly regulated developmental program. The plant vasculature is a complex tissue system consisting of xylem and phloem tissues with a layer of cambial cells in between. Multiple regulatory steps are involved in vascular development. Although molecular and genetic studies have uncovered a variety of key factors controlling vascular development, studies of the actual functions of these factors have been limited due to the inaccessibility of the plant vasculature. Thus, to obtain a different perspective, culture systems have been widely used to analyze the sequential processes that occur during vascular development. A tissue culture system known as VISUAL, in which molecular genetic analysis can easily be performed, was recently established in Arabidopsis thaliana. This reconstitutive approach to vascular development enables this process to be investigated quickly and easily. In this review, I summarize our recent knowledge of the regulatory mechanisms underlying vascular development and provide future perspectives on vascular analyses that can be performed using VISUAL.

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Role of auxin and cytokinins in vascular cambium or callus formation - Biology

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Endogenous auxin biosynthesis and de novo root organogenesis Ya Lin Sang, Zhi Juan Cheng and Xian Sheng Zhang* State Key Laboratory of Crop Biology, College of Life Sciences, College of Forestry, Shandong Agricultural University, Taian, Shandong 271018, China * Correspondence: [email protected]

Induction of adventitious roots is essential for vegetative propagation of plants, and auxin has long been used as an exogenous root-inducing agent. In this issue of Journal of Experimental Botany, Chen et al. (pages 4273–4284) demonstrate that different members of the YUCCA family orchestrate the endogenous auxin biosynthesis that is required for the induction of adventitious roots.

Sun Wukong, also known as the Monkey King, is the main character in the classical Chinese novel Journey to the West. As a fabled deity, he was endowed with magical properties allowing each of his hairs to be transformed into clones of himself as needed. Plants also possess the remarkable ability of multiplication, with detached pieces of adult tissues capable of forming an entire plant body (Gordon et al., 2007 Birnbaum and Sánchez Alvarado, 2008 Sugimoto et al., 2010). This unique ability is mainly based on de novo organogenesis, in which adventitious

shoots or roots are generated from isolated tissues or organs (Duclercq et al., 2011 Cheng et al., 2013 Xu and Huang, 2014). De novo organogenesis can be induced under both natural and tissue culture conditions (Chen et al., 2014). Plant organs, such as stems and leaves, give rise to adventitious roots under natural growth conditions and this property has long been used for vegetative propagation of elite genotypes in agriculture, forestry and horticulture (De Klerk et al., 1999). Six decades ago, Skoog and Miller demonstrated that the entire plant could be regenerated by tissue culture (Skoog and Miller, 1957). They showed that culturing explants in medium containing high levels of cytokinin induced the formation of adventitious shoots, whereas medium with high levels of auxin triggered initiation of adventitious roots. This classic system laid the foundations for plant micropropagation and genetic transformation (Duclercq et al., 2011 Li et al., 2011). In both cases, a key step ensuring the success of plant regeneration is de novo root organogenesis,

© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

4012 | Box 1. Modulation of the dynamic expression pattern of YUC4 in response to wounding After detachment, leaf explants were cultured on B5 medium without exogenous hormone. At day 0, YUC4 is expressed in the hydathode. During the formation of adventitious roots, its expression is enhanced in mesophyll cells. After two days, strong expression signals are detected in the vascular tissues near the wound, where the adventitious roots initiate. DAC, days after culture.

which guarantees the water and nutrient supply for regeneration and survival of the new organism (Chen et al., 2014).

Cell fate transition The first cellular event of de novo organogenesis is cell fate transition (Duclercq et al., 2011 Chatfield et al., 2013). Evidence from different species has indicated that adventitious roots initiate from the procambium or cambium regions (Liu et al., 2014 Xu and Huang, 2014). To study the underlying mechanisms, Chen et al. have developed a simple system to mimic the formation of adventitious roots under natural conditions (Chen et al., 2014). By culturing Arabidopsis leaves on B5 medium without exogenous hormones for six to eight days, adventitious roots can be generated from the midvein near the wound (Liu et al., 2014). Using this system, Liu et al. revealed that the cell fate transition during the initiation of rooting contains two steps. In the first step, WUSCHELRELATED HOMEOBOX 11 (WOX11) and WOX12 act redundantly to regulate the transition from procambium cells to root founder cells. In the second step, root founder cells are further switched into root primordium cells, marked by WOX5 expression. Notably, endogenous auxin plays essential roles in the cell fate reprogramming process. Inhibiting polar auxin transport using naphthylphthalamic acid (NPA) abolishes rooting, an effect which can be rescued by exogenous indole-3-acetic acid. During adventitious root formation, the distribution of auxin response signals overlaps with the expression regions of WOX11. Mutations of the auxin response elements within the WOX11 promoter or NPA treatment disrupt the expression pattern of WOX11 (Liu et al., 2014). Moreover, the auxin response signals are progressively enhanced and distributed in the region of root initiation, suggesting that the wounding induces auxin accumulation in this area. However, the molecular events between explant detachment and adventitious root initiation remain to be elucidated.

YUCCA enzymes The research reported in this issue by Chen et al. (2016) describes the involvement of different members of the

YUCCA (YUC) family, which encode enzymes catalysing the rate-limiting step of auxin biosynthesis in de novo root organogenesis. Under natural growth conditions, some plant species can generate adventitious roots from detached organs spontaneously, but in most cases application of exogenous auxin is required (De Klerk et al., 1999), suggesting that the endogenous auxin biosynthesis varies among the different species. However, using culture methods to induce rooting using exogenous auxin could bypass the function of endogenous hormones. In the system used by Chen et al. (2016), no exogenous hormones were added. Thus, adventitious root generation depended on endogenous hormones, providing an opportunity to investigate the roles of endogenous auxin in de novo root organogenesis. Together with the previous findings from this lab (Chen et al., 2014 Liu et al., 2014), the results support a working model for de novo root organogenesis (see Chen et al., 2016, Fig. 9). The detachment of leaf explants leads to significant increases in the level of auxin. The elevated auxin content results from the function of YUC genes, which respond to wounding and act upstream of cell fate transition from competent cells (procambium and vascular parenchyma cells) to root founder cells. The YUC genes show division of labour and orchestrate auxin biosynthesis required for the formation of adventitious roots. Of them, YUC1, 2, 4 and 6 play major roles under both light and dark conditions. YUC1 and 4 function in a wounding-induced way, whereas YUC2 and 6 contribute to the basal auxin level (Box 1). In addition, YUC5, 8 and 9 mainly produce auxin in leaf margin and mesophyll cells in response to darkness.

More questions A critical open question is how wounding signals trigger the spatial expression of YUC1 and 4. The authors suggest that wounding-response factors could regulate YUC1 and 4 in cooperation with epigenetic factors. This is supported by the fact that up-regulation of YUC1 and 4 expression is accompanied by a reduced level of histone H3 lysine 27 trimethylation, a marker of transcriptional repression (Schatlowski et al., 2008 He et al., 2013). It would be interesting to investigate the relationship between wounding signals and epigenetic factors, as well as their regulatory role in de novo root organogenesis.

| 4013 Despite its importance to plant industries worldwide, adventitious root induction is still difficult in many species, hampering the development of forestry and horticulture (Rasmussen et al., 2012). The issue is mainly limited knowledge about the mechanisms controlling adventitious root formation, and therefore the findings presented here provide valuable new information. Genetic engineering approaches allowing the modification of endogenous auxin biosynthesis would now be powerful in enhancing our abilities.

cytokinin biosynthesis by AUXIN RESPONSE FACTOR3. Plant Physiology 161, 240–251.

Key words: Adventitious root, auxin biogenesis, de novo organogenesis, plant regeneration, YUCCA family.

He C, Huang H, Xu L. 2013. Mechanisms guiding Polycomb activities during gene silencing in Arabidopsis thaliana. Frontiers in Plant Science 4, 454.

Journal of Experimental Botany, Vol. 67 No. 14 pp. 4011–4013, 2016 doi:10.1093/jxb/erw250

References Birnbaum KD, Sanchez Alvarado A. 2008. Slicing across kingdoms: regeneration in plants and animals. Cell 132, 697–710. Chatfield SP, Capron R, Severino A, Penttila PA, Alfred S, Nahal H, Provart NJ. 2013. Incipient stem cell niche conversion in tissue culture: using a systems approach to probe early events in WUSCHEL-dependent conversion of lateral root primordia into shoot meristems. The Plant Journal 73, 798–813. Chen L, Tong J, Xiao L, Ruan Y, Liu J, Zeng M, Huang H, Wang J-W, Xu L. 2016. YUCCA-mediated auxin biogenesis is required for cell fate transition occurring during de novo root organogenesis in Arabidopsis. Journal of Experimental Botany 67, 4273–4284.

De Klerk G, VanDerKrieken W, DeJong J. 1999. The formation of adventitious roots: new concepts, new possibilities. In Vitro Cellular & Developmental Biology – Plant 35, 189–199. Duclercq J, Sangwan-Norreel B, Catterou M, Sangwan RS. 2011. De novo shoot organogenesis: from art to science. Trends in Plant Science 16, 597–606. Gordon SP, Heisler MG, Reddy GV, Ohno C, Das P, Meyerowitz EM. 2007. Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development 134, 3539–3548.

Li W, Liu H, Cheng ZJ, Su YH, Han HN, Zhang Y, Zhang XS. 2011. DNA methylation and histone modifications regulate de novo shoot regeneration in Arabidopsis by modulating WUSCHEL expression and auxin signaling. PLOS Genetics 7, e1002243. Liu J, Sheng L, Xu Y, Li J, Yang Z, Huang H, Xu L. 2014. WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis. The Plant Cell 26, 1081–1093. Rasmussen A, Mason MG, De Cuyper C, et al. 2012. Strigolactones suppress adventitious rooting in Arabidopsis and pea. Plant Physiology 158, 1976–1987. Schatlowski N, Creasey K, Goodrich J, Schubert D. 2008. Keeping plants in shape: polycomb-group genes and histone methylation. Seminars in Cell & Developmental Biology 19, 547–553. Skoog F, Miller CO. 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symposia of the Society for Experimental Biology 11, 118–130.

Chen X, Qu Y, Sheng L, Liu J, Huang H, Xu L. 2014. A simple method suitable to study de novo root organogenesis. Frontiers in Plant Science 5, 208.

Sugimoto K, Jiao Y, Meyerowitz EM. 2010. Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Developmental Cell 18, 463–471.

Cheng ZJ, Wang L, Sun W, et al. 2013. Pattern of auxin and cytokinin responses for shoot meristem induction results from the regulation of

Xu L, Huang H. 2014. Genetic and epigenetic controls of plant regeneration. Current Topics in Developmental Biology 108, 1–33.


One per cent for Fringe Scientists

Die Zeit July 11, 2002, page 28
Interview with Rupert Sheldrake

Rupert Sheldrake wants the public to share in decisions about allocation of research funds

Die Zeit: Many surveys show that science is losing its trust with the public, be it food safety or the vaccination against measles and mumps in England. What is your suggestion on how to close this gap in confidence?

Rupert Sheldrake: Mainstream science sees this as an image crisis that can be overcome by a better public understanding of science. But this crisis runs deeper. Citizens are alienated from scientific institutions that should actually serve them and who are paid by them.

Die Zeit: Where does this alienation originate?

Sheldrake: Science has developed into a bureaucratic, rigid system. Receiving research funds, publishing papers, being promoted and getting prestige - all this depends on the peer reviews of anonymous committees. This reinforces current opinions .

Die Zeit: . but at the same time it serves to reject low-quality research and undesirable trends.

Sheldrake: The established system may prevent stupid research but it also slows down originality and innovation, promotes timidness and conformity. Innovation, however, is absolutely necessary in science.

Die Zeit: Was it any different in the past?

Sheldrake: At least in the USA and in England science was less institutionalised in the 19th century. A scientist like Darwin, who held no academic position and received no public funds, would probably not have been able to do his research on evolution under today‘s circumstances. Important breakthroughs back then were mostly produced by researchers who were neither professional scientists nor part of a bureaucratic system.

Die Zeit: Does only research suffer from this?

Sheldrake: No, we all pay a high price for it. Research uses up large amounts of money but often serves neither the public interest nor does it produce innovation. For example 50 per cent of all the scientific papers are not read by anyone except the authors themselves.

Die Zeit: How do new insights get lost this way?

Sheldrake: This can be seen very clearly in the field of alternative medicine. What is supported are mainly conventional projects, like the genome project or molecular diagnosis. I do not mean to say that this is worthless. But in this way only a fraction of the medical problems is dealt with. At the same time millions of people seem to benefit from acupuncture, herbs and other therapies. These low-cost forms of therapy, however, are largely ignored by the official bodies because they do not fit the dominating paradigm of mechanistic biology.

Die Zeit: How might things be done differently?

Sheldrake: In the USA senators and congressmen have succeeded in establishing a Center for Alternative Medicine with an initial annual budget of one million dollars, against the fierce resistance of the scientific establishment that spoke of squandering and quackery. But the research results were so promising that the budget has now been increased to 100 million dollars. Even that is still minimal compared to the many billions spent on conventional research.

Die Zeit: So you are asking for a more democratic decision-making?

Sheldrake: Research should reflect the interests of the citizen and taxpayer. At the present the main part of the funds is distributed by the scientific establishment as it sees fit, controlled by politicians. But politicians trust their advisers, and these are from the establishment again. A little democratisation might serve as a corrective here.

Die Zeit: And how would that work out in detail?

Sheldrake: My suggestion is to use one per cent of all research funding on projects that public interest decides about. 99 per cent would be distributed as before, by means of small committees which I call the College of Cardinals. I do not suppose that the official bodies will give up their control of research budgets, which is only human and understandable.

Die Zeit: What is your defintion of „public interest“?

Sheldrake: It might be best to use techniques of opinion research, like polls or focus groups, to find out what people want.

Die Zeit: Maybe they don‘t know what they want.

Sheldrake: Most of them do have clear ideas. Alternative research in the USA only received more grants because there was a keen public interest, which politicians took up. Surveys also show that there is an urgent desire for more and independent research on food safety.

Die Zeit: How about decision by lots?

Sheldrake: No problem. It would be worth an empirical test. 80 per cent of the "alternative“ budget might be allocated democratically and the remaining 20 per cent might be subjected to the chance of dice. After five years the results might be assessed by an independent body. This would show whether chance or democracy lead to better results.

Die Zeit: Which body would you want to make the decisons about research applications?

Sheldrake: One possibility would be committees, another one would be representatives of NGOs. Some kind of democratic National Council for Science might be established, or some society, like the Royal Geographical Society, might get the mandate.

Die Zeit: Who is to apply?

Sheldrake: Certainly also amateur scientists. Our societies are much more educated and better trained than at Darwin‘s time.The internet allows access to information which in former days was accessible only to people with large libraries. There are ideal conditions for liberating scientific research from bureaucratic institutions.

Die Zeit: People might accuse you of making this proposal just to serve your personal interests, like the studies of parapsychological phenomena, that are not funded publicly.

Sheldrake: I do not get any public funds for my research, nor am I striving for any. I am fortunate enough to receive grants from private foundations.

Die Zeit: Are there also mainstream scientists who are warming to your ideas?

Sheldrake: Many colleagues are conscious of the pressure that the present system produces. Part of it goes back to the negative influence of the Citations Index. The allocation of research grants does not depend on the quality of the work but on how often it is quoted. This supports today‘s majority views and leads to an unhealthy narrowing. If someone works in a new field it is only natural that he is not quoted frequently, since there are few people who know the field of study well enough. Even representatives of the establishment are well aware of this danger, and many of my colleagues encourage me in private.

Die Zeit: If the success of publicly supported projects had to be judged by their practical usefulness, your research on unusual abilities of dogs would not pass the test.

Sheldrake: Materialistic criteria should not be the measure of all things. Public interest should also be a criterion. Many people are fascinated by research on animal behaviour, as the ratings of films about nature show all over the world. But funds are almost only available for the study of the genome of songbirds, not the study of their behaviour.

Die Zeit: You have been working outside the institutions for 20 years. Why this concern about science now?

Sheldrake: Because the crisis has become more fundamental. My suggestions might help to bring life into rigid conditions without turning them upside down. Interests and institutions would remain intact but there is a chance to open new fields to research.

Die Zeit: How do you want to start the process?

Sheldrake: First of all a wide debate is needed. Maybe others have better ideas.

Die Zeit: Is there a lack of public debate in the field of science?

Sheldrake: Very clearly so! Right now there is no forum for it. Anyone who has differing opinions has a hard time. Disputations in the middle ages always held a role for the advocatus diaboli. Thomas Aquinas, one of the great scholars, wrote works in the style of debates in which the arguments of both sides were developed. We can learn a lot from these classical formats. What we have instead is an infinite number of scientific papers carrying the stamp of being officially accepted.

Interview by Jürgen Krönig.

Translated by Helmut Lasarcyk