Regulation of Vascular Development by CLE Peptide-receptor Systems

Cell division and differentiation of stem cells are controlled by non-cell-autonomous signals in higher organisms. The plant vascular meristem is a stem-cell tissue comprising procambial cells that produce xylem cells on one side and phloem cells on the other side. Recent studies have revealed that TDIF (tracheary element differentiation inhibitory factor)/CLE41/CLE44 peptide signal controls the procambial cell fate in a non-cell-autonomous manner. TDIF produced in and secreted from phloem cells is perceived by TDR/PXY, a leucine-rich repeat receptor kinase located in the plasma membrane of procambial cells. This signal suppresses xylem cell differentiation of procambial cells and promotes their proliferation. In addition to TDIF, some other CLE peptides play roles in vascular development. Here, we summarize recent advances in CLE signaling governing vascular development.     

A novel function of TDIF-related peptides: promotion of axillary bud formation

Small peptides derived from the CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) gene family play a key role in various cell-cell communications in land plants. Among them, tracheary element differentiation inhibition factor (TDIF; CLE41/CLE44 peptide) and CLE42 peptide of Arabidopsis have almost identical amino acid sequences and act as inhibitors of tracheary element differentiation. In this study, we report a novel function of TDIF and CLE42. We found by the GUS (β-glucuronidase) reporter gene assay that while CLE41 and CLE44 are expressed preferentially in vascular bundles, CLE42 is expressed strongly in the shoot apical meristem (SAM) and axillary meristems. Overexpression of CLE42 and CLE41 enhanced axillary bud formation in the leaf and cotyledon axils. Before floral transition, the emergence of axillary buds in these plants occurred in an acropetal order. Exogenous supply of either TDIF or CLE42 peptide to the wild type induced similar excess bud emergence. In vascular bundles, the TDIF RECEPTOR (TDR) acts as the main receptor for TDIF. The axillary bud emergence of tdr mutants was little affected by either of the peptides. It was confirmed by scanning electron microscopy that peptide-treated wild-type plants form an axillary meristem-like structure earlier than non-treated plants. SHOOT MERISTEMLESS (STM), a marker gene for meristems, was up-regulated in peptide-treated plants before the axillary meristem becomes morphologically distinguishable. These results indicate that CLE42 peptide and TDIF have an activity to enhance axillary bud formation via the TDR. Judging from its expression pattern, CLE42 may play an important role in the regulation of secondary shoot development.     

TDIF Peptide Signaling Regulates Vascular Stem Cell Proliferation via the WOX4 Homeobox Gene in Arabidopsis

The indeterminate nature of plant growth and development depends on the stem cell system found in meristems. The Arabidopsis thaliana vascular meristem includes procambium and cambium. In these tissues, cell–cell signaling, mediated by a ligand-receptor pair made of the TDIF (for tracheary element differentiation inhibitory factor) peptide and the TDR/PXY (for TDIF RECEPTOR/ PHLOEM INTERCALATED WITH XYLEM) membrane protein kinase, promotes proliferation of procambial cells and suppresses their xylem differentiation. Here, we report that a WUSCHEL-related HOMEOBOX gene, WOX4, is a key target of the TDIF signaling pathway. WOX4 is expressed preferentially in the procambium and cambium, and its expression level was upregulated upon application of TDIF in a TDR-dependent manner. Genetic analyses showed that WOX4 is required for promoting the proliferation of procambial/cambial stem cells but not for repressing their commitment to xylem differentiation in response to the TDIF signal. Thus, at least two intracellular signaling pathways that diverge after TDIF recognition by TDR might regulate independently the behavior of vascular stem cells. Detailed observations in loss-of-function mutants revealed that TDIF-TDR-WOX4 signaling plays a crucial role in the maintenance of the vascular meristem organization during secondary growth.     

CLE Peptides in Vascular Development

[ Guodong Wang (Corresponding author)] The plant vascular system consists of two conductive tissues, phloem and xylem. The vascular meristem, namely the (pro‐)cambium, is a stem‐cell tissue that gives rise to both xylem and phloem. Recent studies have revealed that CLAVATA3/Embryo Surrounding Region‐related (CLE) peptides function in establishing the vascular system through interaction with phytohormones. In particular, TDIF/CLE41/CLE44, phloem‐derived CLE peptides, promote the proliferation of vascular cambium cells and prevent them from differentiating into xylem by regulating WOX4 expression through the TDR/PXY receptor. In this review article, we outline recent advances on how CLE peptides function in vascular development in concert with phytohormones through mediating cell‐cell communication. The perspective of CLE peptide signaling in vascular development is also discussed.     

CLE25 peptide regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex

The phloem, located within the vascular system, is critical for delivery of nutrients and signaling molecules throughout the plant body. Although the morphological process and several factors regulating phloem differentiation have been reported, the molecular mechanism underlying its initiation remains largely unknown. Here, we report that the small peptide‐coding gene, CLAVATA 3 (CLV3)/EMBEYO SURROUNDING REGION 25 (CLE25), the expression of which begins in provascular initial cells of 64‐cell‐staged embryos, and continues in sieve element‐procambium stem cells and phloem lineage cells, during post‐embryonic root development, facilitates phloem initiation in Arabidopsis. Knockout of CLE25 led to delayed protophloem formation, and in situ expression of an antagonistic CLE25_G6T peptide compromised the fate‐determining periclinal division of the sieve element precursor cell and the continuity of the phloem in roots. In stems of CLE25_G6T plants the phloem formation was also compromised, and procambial cells were over‐accumulated. Genetic and biochemical analyses indicated that a complex, consisting of the CLE‐RESISTANT RECEPTOR KINASE (CLERK) leucine‐rich repeat (LRR) receptor kinase and the CLV2 LRR receptor‐like protein, is involved in perceiving the CLE25 peptide. Similar to CLE25, CLERK was also expressed during early embryogenesis. Taken together, our findings suggest that CLE25 regulates phloem initiation in Arabidopsis through a CLERK‐CLV2 receptor complex.     

Plant development: PXY and polar cell division in the procambium

The vascular stem-cell tissue known as procambium generates phloem cells on one side and xylem cells on the other. The Arabidopsis PXY gene encodes a leucine-rich repeat receptor-like kinase that is required for polar divisions of procambial cells.     

Cell differentiation in the longitudinal veins and formation of commissural veins in rice (Oryza sativa) and maize (Zea mays)

Vascular development is a central theme in plant science. However, little is known about the mechanism of vascular development in monocotyledons (compared with dicotyledons). Therefore, we investigated sequential processes of differentiation into various different vascular cells by carrying out detailed observations using serial sections of the bases of developing leaves of rice and maize. The developmental process of the longitudinal vascular bundles was divided into six stages in rice and five stages in maize. The initiation of differentiation into procambial progenitor cells forming the commissural vein arose in a circular layer cell that was adjacent to both a metaxylem vessel and one or a few phloem cells in stage V longitudinal vascular bundles. In most cases the differentiation of ground meristem cells into procambial progenitor cells extended in one direction, toward the next longitudinal vascular bundle, and subsequent periclinal divisions and further differentiation produced a vessel element, two companion cells and a sieve element to form a commissural vein. These results suggest the presence of an intercellular signal(s) that induces differentiation of the circular layer cell and the ground meristem cells into procambial progenitor cells, forming a commissural vein sequentially.     

Peptide signaling in vascular development

In plants and animals, putative small peptide ligands have been suggested to play crucial roles in development as signal molecules of cell-cell communication. Recent studies of CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) genes and their products have revealed that distinctive dodeca-CLE peptide ligands function in various developmental processes. In particular, the finding and characterization of TDIF, a dodeca-CLE peptide suppressing tracheary element differentiation, indicates regulation of vascular organization by cell-cell communication through CLE peptides. In addition, other extracellular peptides such as phytosulfokine, proteins such as xylogen, and phytohormones all participate in the ordered formation of vascular tissues.     

DIFFERENTIATION AND CONTINUITY OF THE PHLOEM IN THE LEAF INTERCALARY MERISTEM OF LOLIUM PERENNE

Serial transverse sections of the stem tip and leaf bases of immature perennial ryegrass leaves were examined to assess the effect of the intercalary meristem on assimilate movement. The development of procambial strands in very young leaves was followed, and the subsequent differentiation of proto- and metaphloem examined in both lamina and sheath. In major bundles the first two or three series of protophloem cells differentiated acropetally into the leaf and they or their crushed remnants could be recognized at all levels. A further three or four series of protophloem cells differentiated basipetally, as did the protophloem of all minor bundles and the metaphloem. The basipetal differentiation of the bulk of the phloem combined with crushing of older cells resulted in acute constriction or even complete local blockage of the functional phloem in the active meristematic region. This constriction of the phloem would tend to divert assimilates into the dividing cells both from the stem below and from the mature upper portion of the leaf. No constriction was found at the base of a mature sheath.     

Protophloem differentiation in early Arabidopsis thaliana development

During Arabidopsis embryogenesis, procambial cells undergo coordinated, asymmetric cell divisions, giving rise to vascular precursor cells (protophloem and protoxylem precursors). After germination, these cells terminally differentiate into specialized conducting cells, referred to as protophloem and protoxylem cells. Few readily identifiable markers of the onset of specification and differentiation are available, hampering the molecular genetic analysis of protophloem development. Confocal microscopy was used to investigate the patterning and differentiation of phloem cells during early plant development. Longitudinal divisions of phloem initials allowed the identification of protophloem precursor cells and adjacent metaphloem initials along the length of the plant. During germination, protophloem differentiation was observed at two independent locations, in the cotyledons and the hypocotyl. In both locations, differentiation was concomitant with cell elongation. We identified five gene-trap lines (PD1-PD5) with marker gene expression in immature protophloem elements. The spatio-temporal marker expression pattern of the lines divides them into two groups. The early specification markers PD4 and PD5 were expressed in developing organs before procambium formation and then became restricted to phloem initial cells. The protophloem precursor markers PD1-PD3 were expressed in differentiating protophloem cells at different stages of their development. All markers were expressed transiently and iteratively during the differentiation of protophloem in newly formed organs. Flanking genes were identified for four out of five gene-trap insertion lines. The possible function of these genes with respect to phloem differentiation is discussed.     

Competitive action between Brassinosteroid and tracheary element differentiation inhibitory factor in controlling xylem cell differentiation

For permanent secondary growth in plants, cell proliferation and differentiation should be strictly controlled in the vascular meristem consisting of (pro)cambial cells. A peptide hormone tracheary element differentiation inhibitory factor (TDIF) functions to inhibit xylem differentiation, while a plant hormone brassinosteroid (BR) promotes xylem differentiation in (pro)cambial cells. However, it remains unclear how TDIF and BR cooperate to regulate xylem differentiation for the proper maintenance of the vascular meristem. In this study, I developed an easy evaluation method for xylem differentiation frequency in a vascular induction system Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL) by utilizing a xylem-specific luciferase reporter line. In this quantitative system, TDIF suppressed and BR promoted xylem differentiation in a dose-dependent manner, respectively. Moreover, simultaneous treatment of TDIF and BR with (pro)cambial cells revealed that they can cancel their each other’s effect on xylem differentiation, suggesting a competitive relationship between TDIF and BR. Thus, mutual inhibition of “ON” and “OFF” signal enables the fine-tuned regulation of xylem differentiation in the vascular meristem.     

木立芦荟叶的发育解剖学研究

应用植物解剖学方法研究了木立芦荟（Aloe arborescens Mill.)叶的发育过程。研究结果表明,叶原基在发育早期其形态是不对称的,内部为同形细胞组成,但很快分化成原表皮,原形成层束和基本分生组织。以后,原表皮发育成表皮,位于原表皮下的2－5层基本分生组织细胞发民同化薄壁组织,而位于中央的基本分生组织细胞则发育成储水薄壁组织,原形成层束发育成维管束。维管束由维管束鞘、木质部、韧皮部和大型薄壁细胞组成。大型薄壁细胞起源于原形成层束,位于韧皮部内,其发育迟于筛管、伴胞,为芦荟属植物叶的结构特征。     

Early Development and Ultrastructure of the Major Veins and Their Sheath Tissues in the Maize Leaves

The report described the ultrastructural changes that occurred in the major veins and their associated bundle sheaths (BS) of the maize ( Zea mays L. ) leaf blade in the process of their differentiation from three adjacent cells in the middle layer of the ground meristem, the minimal number of cells involved with the initiation of a procambial strand and the associated BS. The inner cell underwent two successive unequal periclinal divisions: a smaller cell that later differentiated into the adaxial BS cell precursor, and a larger one that divided once again periclinally yielding an abaxial BS cell precursor and a centrally located procambial initial cell. One of the two lateral cells immediately adjacent to either side of the inner cell also divided periclinally; these derivatives, along with another lateral cell of the original three-celled unit formed the precursor cells of the lateral BS. Prior to the initiation of protophlcem differentiation, all of the procambial cells showed ultrastructural characteristics basically similar to the procambial initial. They possessed a prominent nucleus with electron-dense aggregates of heterochromatin, a dense cytoplasm rich in ribosomes, proplastids and mitochondria; also a thin wall containing numerous plasmodesmata. In many cases, only short pieces of rough endoplasmic reticulum cistemae and a few small sized vacuoles were present. In adclifton, evidence of cytoplasmic disintegration leading to new vacuole formation was noted in the process of proeambium development. It was observed that certain endoplasmic reticulum was engaged in the sequestration and lysis of cytoplasm. No apparent uhrastmctuml difference was found between the BS cell precursors and the procambial initials, that was, the distinction between the procambium and the surrounding BS cells occurred gradually after vein initiation, The major ultrastmctural changes which occurred during the differentiation of the meristematic BS cells into the vacuolated cells were (1) a proplastid to chloroplast transformation going through a prolamellar body stage, and (2) the appearance of the multi-concentric membrane complex which might play a role in the degradation of some ribosomes and other cytoplasmic components during the differentiation of BS cells.     

Primary Phloem Development in the Shoot Apex of Rhizophora mangle L. (Rhizophoraceae)

The primary phloem in the shoot apex of the mangrove Rhizophora mangle L. is largely confined to the comparatively condensed area between the first three leaf pairs. The main extension zone, surrounded by the stipular sheath of the third leaf pair, contains vascular bundles arranged in a procambial ring and characterized by a well-developed primary phloem and a less advanced xylem. The phloem consists of a great number of sieve elements, an equal number of associated companion cells, and a few phloem-parenchyma cells. The differentiation of the sieve-element protoplast (with e.g., chromatolytic nuclear degeneration, loss of the vacuole and most organelles) proceeds largely according to a well-known pattern. Their P-type plastids, however, form their protein crystals rather late and therefore cannot be used as an early cell marker. Lateral sieve-element walls are distinct from other wall parts and walls of other cells by their heavy nacreous thickenings, the formation of which is shown to be strictly correlated with the occurrence and orderly arrangement of cortical microtubules.     

Growth, development, and systematics of ferns: Does Botrychium s.l. (Ophioglossales) really produce secondary xylem?1

Developmental morphology and anatomy of Botrychium s.l. were studied to clarify rhizome ontogeny and patterns of tissue maturation that can be used to test the hypothesis that ferns of the Ophioglossales may represent living progymnosperms. Serial anatomical sections of the rhizomes of B. virginianum and B. dissectum reveal that apical meristematic activity and vascular tissue maturation occur over an extended period of several years and then stop. Most of the xylem consists of radial rows of tracheids and interspersed ray-like xylem parenchyma cells that are similar in these respects to secondary xylem, but pits occur on all tracheid walls as is characteristic of primary xylem. No vascular cambium is initiated in mature primary tissues, nor is there secondary phloem. Radial rows of xylem cells are produced by the direct continuation of divisions that begin at the shoot apical meristem, forming a cylinder of radially aligned procambial cells before the differentiation of protoxylem. Continuing divisions over a period of several years increase the number of thin-walled cells and tracheids in each radial row back to about one internode behind where the current year's frond trace diverges from the rhizome stele. At more proximal levels of the rhizome, procambial cell divisions cease and there is no additional tracheid differentiation. These data reveal that the rhizome matures over an exceedingly long period of several years, but that growth is ultimately determinate, thus supporting hypotheses that the Ophioglossales is more closely related to other groups of living ferns than to progymnosperms and seed plants.     

管花肉苁蓉茎异常结构的发育解剖学研究

管花肉苁蓉茎内存在类似于单子叶植物的散生初生维管束。它由散生在基本分生组织中的原形成层束分化而成。在原形成层来分化的过程中,每个原形成层束可通过分离形成2—7个初生维管束,使初生维管束的数目迅速增加。当初生维管束开始正常次生生长时,正常维管束韧皮部外方的薄壁组织细胞或远离维管柬的薄壁组织细胞转变为异常形成层束。异常次生维管束与正常维管束以韧皮部相对或韧皮部并列的方式排列,或异常次生维管束单个存在于薄壁组织中。