The gapped xylem mutant identifies a common regulatory step in secondary cell wall deposition

The phenotype of the novel gapped xylem (gpx) mutant is described. gpx plants exhibit gaps in the xylem in positions where xylem elements would normally be located. These gaps are not part of the transpiration stream and result in gpx plants having fewer functional xylem elements. The gaps are due to the absence of a secondary cell wall in developing xylem elements, resulting in complete degradation of these elements during cell death, and illustrate the importance of the secondary cell wall in retaining a functional xylem element following programmed cell death. Consequently the gpx phenotype suggests that the processes of secondary cell wall formation and cell death are independently regulated in developing xylem. gpx plants also exhibit a highly irregular pattern of secondary cell wall thickening in interfascicular cells, with some cells apparently undergoing little or no secondary cell wall deposition. Secondary cell wall deposition in plants involves the co-ordinate regulation of several complex metabolic pathways. The gpx mutant identifies a key step involved in regulating the deposition of secondary cell wall material in both xylem and interfascicular cells, and suggests that a common regulatory step controls secondary cell wall formation in these diverse cell types. The gpx mutant offers a unique opportunity to elucidate the mechanism by which the complex processes involved in secondary cell wall formation are co-ordinately regulated.     

ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death

Cell size and secondary cell wall patterning are crucial for the proper functioning of xylem vessel elements in the vascular tissues of plants. Through detailed anatomical characterization of Arabidopsis thaliana hypocotyls, we observed that mutations in the putative spermine biosynthetic gene ACL5 severely affected xylem specification: the xylem vessel elements of the acl5 mutant were small and mainly of the spiral type, and the normally predominant pitted vessels as well as the xylem fibers were completely missing. The cell-specific expression of ACL5 in the early developing vessel elements, as detected by in situ hybridization and reporter gene analyses, suggested that the observed xylem vessel defects were caused directly by the acl5 mutation. Exogenous spermine prolonged xylem element differentiation and stimulated cell expansion and cell wall elaboration in xylogenic cell cultures of Zinnia elegans, suggesting that ACL5 prevents premature death of the developing vessel elements to allow complete expansion and secondary cell wall patterning. This was further supported by our observations that the vessel elements of acl5 seemed to initiate the cell death program too early and that the xylem defects associated with acl5 could be largely phenocopied by induction of premature, diphtheria toxin-mediated cell death in the ACL5-expressing vessel elements. We therefore provide, for the first time, mechanistic evidence for the function of ACL5 in xylem specification through its action on the duration of xylem element differentiation.     

Programmed cell death of plant tracheary elements differentiating in vitro

Summary We used various microscopic and labeling techniques to examine events occurring during the programmed cell death (PCD) of plant tracheary elements (TEs) developing in vitro. TEs differentiating in vitro synthesize a secondary cell wall which is complex in composition and pattern at approximately 72 h after hormone manipulation. The timing of PCD events was established relative to this developmental marker. Cytoplasmic streaming continues throughout secondary wall synthesis, which takes 6 h to complete in a typical cell. Vital dye staining and ultrastructural analysis show that the vacuole and plasma membrane are intact during secondary cell wall synthesis, but the cytoplasm becomes less dense in appearance, most likely through the action of confined hydrolysis by small vacuoles which are seen throughout the cell at this time. The final, preeminent step of TE PCD is a rapid collapse of the vacuole occurring after completion of secondary cell wall synthesis. Vacuole collapse is an irreversible commitment to death which results in the immediate cessation of cytoplasmic streaming and leads to the complete degradation of cellular contents, which is probably accomplished by release of hydrolytic enzymes sequestered in the vacuole. This event represents a novel form of PCD. The degradation of nuclear DNA is detectable by TUNEL, an in situ labeling method, and appears to occur near or after vacuole collapse. Our observations indicate that the process of cellular degradation that produces the hollow TE cell corpse is an active and cell-autonomous process which is distinguishable morphologically and kinetically from necrosis. Although TE PCD does not resemble apoptosis morphologically, we describe the production of spherical protoplast fragments by cultured cells that resemble apoptotic bodies but which are not involved in TE PCD. We also present evidence that, unlike the hypersensitive response (HR), TE PCD does not involve an oxidative burst. While this evidence does not exclude a role for reactive oxygen intermediates in TE PCD, it does suggest TE PCD is mechanistically distinct from cell death during the HR.Abbreviations BA 6-benzylamino-purine - DAPI 4,6-diamidino-2-phenylindole diacetate - DCF 2,7-dichlorofluorescein diacetate - DPI diphenyleneiodonium - FDA fluorescein diacetate - HR hypersensitive response - NAA -naphthalene-acetic acid - PCD programmed cell death - ROI reactive oxygen intermediate - TE tracheary element - TUNEL TdT-mediated dUTP nick end labeling     

Tracheary Element Differentiation Uses a Novel Mechanism Coordinating Programmed Cell Death and Secondary Cell Wall Synthesis

Tracheary element differentiation requires strict coordination of secondary cell wall synthesis and programmed cell death (PCD) to produce a functional cell corpse. The execution of cell death involves an influx of Ca²⁺ into the cell and is manifested by rapid collapse of the large hydrolytic vacuole and cessation of cytoplasmic streaming. This precise means of effecting cell death is a prerequisite for postmortem developmental events, including autolysis and chromatin degradation. A 40-kD serine protease is secreted during secondary cell wall synthesis, which may be the coordinating factor between secondary cell wall synthesis and PCD. Specific proteolysis of the extracellular matrix is necessary and sufficient to trigger Ca²⁺ influx, vacuole collapse, cell death, and chromatin degradation, suggesting that extracellular proteolysis plays a key regulatory role during PCD. We propose a model in which secondary cell wall synthesis and cell death are coordinated by the concomitant secretion of the 40-kD protease and secondary cell wall precursors. Subsequent cell death is triggered by a critical activity of protease or the arrival of substrate signal precursor corresponding with the completion of a functional secondary cell wall.     

杜仲次生木质部分化和脱分化过程中酸性磷酸酶的超微细胞化学定位

杜仲（EucommiaulmoidesOliv．）次生木质部分化过程中,在形成层刚衍生的木薄壁细胞中,酸性磷酸酶（APase）主要分布于核膜边缘和高尔基体;在分化程度较高的木薄壁细胞中,APase散布于整个核中,进而,在各种细胞器残体上聚集;在成熟的木薄壁细胞中,APase沿细胞壁内侧分布。在未成熟导管分子中,核、质膜及纹孔上明显存在APase聚集,进而,核解体;在即将分化成熟的导管分子中,APase主要集中于初生壁;在已分化成熟的导管分子中,APase集中于次生壁。脱分化过程中,只在细胞质中可见分散的APase活性,而细胞核和细胞壁上未见此酶的分布;更深层的即将分化成熟和已分化成熟的导管分子,未见有细胞分裂,其上APase的分布与剥皮前相同。通过比较分化和脱分化过程中APase的分布,推测不同的APase同工酶可能分别参与了次生木质部细胞程序性死亡过程中原生质体的解体和次生壁的建成。APase的聚集程度可能是决定细胞能否脱分化的一个重要特征。     

A Cytochemical Study of Cell Wall Hydrolysis in the Secondary Xylem of Poplar (Populus italica Moench)

Certain developmental features of cell wall hydrolysis werestudied in the secondary xylem of poplar (Populus italica Moench).At the intervascular pit membrane hydrolysis starts prematurelybefore differentiation of the secondary wall is complete andincreases progressively. Eventually the whole of the middlelamella is hydrolysed, and the primary wall undergoes lyticmodification. The modified polysaccharides are dispersed, presumablyby the transpiration stream. During differentiation the vessel-parenchymapit membrane remains unaltered and undergoes thickening. Thepresent investigation suggests that the plasalemma plays animportant role in the ordered hydrolysis of certain regionsof the primary walls. Populus italicaMoench, poplar, secondaryxylem, xylem, cell wall hydrolysis, plasmalemma, pit membram     

Ultrastructural Changes in Cambial Cell Derivatives during Xylem Differentiation in Poplar

Abstract: The changes in cellular structures that occur in cambial cell derivatives during xylogenesis were examined in Populus trichocarpa Torr et Gray. During dormancy, the cells of the vascular cambium are characterised by dense cytoplasm, many small vacuoles and lipid bodies. During cambial activation, cambial cells are highly vacuolated, the cytoplasm is rich in organelles and the nucleus contains distinctly enlarged nucleoli. The plasma membrane forms vesicle-filled invaginations which mediate uptake of vesicular material into the vacuole. The mitotic patterns in dividing fusiform cells are fragmentary due to their strong vacuolisation. During cell enlargement, cambial cell derivatives remain strongly vacuolated and cytoplasmic structures are similar to active fusiform cells. From the beginning of secondary cell wall formation many changes in cytoplasmic structures occur in newly-formed fibres and vessels. In fibres, the cytoplasm is characterised by components of secondary cell wall synthesis, as indicated by increased amounts of endoplasmic reticulum, vesicle-producing dictyosomes and microtubules. In contrast, vessels show a more or less distinct occurrence of these components and remain more strongly vacuolated than fibres. Similar to cambial cells, a distinct flow of vesicular material into the vacuole through invaginations of the plasma membrane is apparent in fibres, as well as in vessels. After completion of the secondary cell walls, the loss of tonoplast integrity causes the collapse of the vacuole and initiates cell death in vessels and fibres. In vessels the tonoplast exhibits unusually strong staining prior to the collapse of the vacuole, indicating subsequent cell death. Overall, our results indicate an important role for the vacuole in the xylogen differentiation of cambial derivatives.     

Role of Arabidopsis RabG3b and autophagy in tracheary element differentiation

The vascular system of plants consists of two conducting tissues, xylem and phloem, which differentiate from procambium cells. Xylem serves as a transporting system for water and signaling molecules and is formed by sequential developmental processes, including cell division/expansion, secondary cell wall deposition, vacuole collapse and programmed cell death (PCD). PCD during xylem differentiation is accomplished by degradation of cytoplasmic constituents, and it is required for the formation of hollow vessels, known as tracheary elements (TEs). Our recent study revealed that the small GTPase RabG3b acts as a regulator of TE differentiation through its autophagic activation. By using an Arabidopsis in vitro cell culture system, we showed that autophagy is activated during TE differentiation. Overexpression of a constitutively active RabG3b (RabG3bCA) significantly enhances both autophagy and TE differentiation, which are consistently suppressed in transgenic plants overexpressing a dominant negative form (RabG3bDN) or RabG3b RNAi (RabG3bRNAi), a brassinosteroid-insensitive mutant bri1-301 and an autophagy mutant atg5-1. On the basis of our results, we propose that RabG3b functions as a component of autophagy and regulates TE differentiation by activating the process of PCD.     

ORGANIZATION AND BREAKDOWN OF THE PROTOPLAST DURING MATURATION OF PINE TRACHEIDS

The protoplast of maturing axial tracheids in the secondary xylem of shortleaf pine (Pinus echinata Mill.) was studied by transmission and scanning electron microscopy. The mature protoplast is differentiated into two interconnected components: (1) the commonly observed peripheral layer lining the secondary cell wall, and (2) an elaborate reticulum of cytoplasmic filaments and placoids within the central vacuole. The reticulum provides an extensive surface area of vacuolar membranes for rapid exchange of nutrients and metabolites with the vacuolar sap, which is envisaged to function as a vital medium during the period of secondary cell wall synthesis. The breakdown of the protoplast which terminates tracheid maturation is associated with poorly defined alterations of the vacuolar membranes. This is indicated by increased formation of cytoplasmic spherules and membraneous vesicles which may be portions of separated vacuolar membrane during early stages of degradation. Autolysis is supposed to occur when the cytoplasm is exposed to the vacuolar sap after rupture and separation of the vacuolar membranes. The Gomori acid phosphatase technique as combined with electron microscopy produced no evidence of autolysosomal segresomes in strands of intravacuolar reticulum of the cytoplasm.     

Three loblolly pine CesA genes expressed in developing xylem are orthologous to secondary cell wall CesA genes of angiosperms

Specific plant cellulose synthases (CesA), encoded by a multigene family, are necessary for secondary wall synthesis in vascular tissues and are critical to wood production. We obtained full-length clones for the three CesAs that are highly expressed in developing xylem and examined their phylogenetic relationships and expression patterns in loblolly pine tissues. Full-length CesA clones were isolated from cDNA of developing loblolly pine (Pinus taeda) xylem and phylogenetic inferences made from plant CesA protein sequences. Expression of the three genes was examined by Northern blot analysis and semiquantitative RT-PCR. Each of three PtCesA genes is orthologous to one of the three angiosperm secondary cell wall CesAs. The PtCesAs are coexpressed in tissues of loblolly pine with tissues undergoing secondary cell wall biosynthesis showing the highest levels of expression. Phylogenetic and expression analyses suggest that functional roles for these loblolly pine CesAs are analogous to those of orthologs in angiosperm taxa. Based upon evidence from this and other studies, we suggest division of seed plant CesA genes into six major paralogous groups, each containing orthologs from various taxa. Available evidence suggests that paralogous CesA genes and their distinct functional roles evolved before the divergence of gymnosperm and angiosperm lineages.     

Changes in phenolic acids during maturation and lignification of scots pine xylem

The content and fractional composition of alcohol soluble phenolic acids (PhA) in cells with different degree maturation and lignification in the course of early and late wood formation in the pine (Pinus sylvestris L.) stem during vegetation were studied. Phenolic compounds (PhC), extracted by 80% ethanol, were divided into free and bound fractions of PhA. In turn, the esters and ethers were isolated from bound PhA. The contents of all substances were calculated per dry weight and per cell. Considerable differences have been found to exist in both the contents and the composition of the fractions PhA on successive stages of tracheid maturation of early and late xylem. Early wood tracheids at all secondary wall thickening steps contained PhC less and free PhA more than late wood tracheids. Throughout earlywood tracheid maturation, the pool of free PhA per cell declined at the beginning of lignification and then increased gradually while that of bound PhA decreased. The maturation of late wood tracheids were accompanied by the rise of free PhA pool and the diminution of bound PhA pool. In the composition of bound PhA, the ethers were always dominant, and the amount of that in earlywood cells was less than in latewood cells. The cells of early xylem at all steps of maturation contained more of esters. The sum total of free hydroxycinnamic acids, precursors of monolignols, gradually decreased during early xylem lignification as the result of the reduction of the pools of p-coumaric, caffeic, ferulic and synapic acids, while that of their esters rised. In the course of late xylem lignification, the pools of free p-coumaric, ferulic and, especially, synapic acids increased. Simultaneously, the amount of ferulic acid ester and synapic acid ether increased too. According to the data, lignin biosynthesis in early xylem and late xylem occurs with different dynamics and the structure of lignins of two xylem types might be different too.     

Perturbation of polyamine catabolism can strongly affect root development and xylem differentiation

Spermidine (Spd) treatment inhibited root cell elongation, promoted deposition of phenolics in cell walls of rhizodermis, xylem elements, and vascular parenchyma, and resulted in a higher number of cells resting in G(1) and G(2) phases in the maize (Zea mays) primary root apex. Furthermore, Spd treatment induced nuclear condensation and DNA fragmentation as well as precocious differentiation and cell death in both early metaxylem and late metaxylem precursors. Treatment with either N-prenylagmatine, a selective inhibitor of polyamine oxidase (PAO) enzyme activity, or N,N(1)-dimethylthiourea, a hydrogen peroxide (H(2)O(2)) scavenger, reverted Spd-induced autofluorescence intensification, DNA fragmentation, inhibition of root cell elongation, as well as reduction of percentage of nuclei in S phase. Transmission electron microscopy showed that N-prenylagmatine inhibited the differentiation of the secondary wall of early and late metaxylem elements, and xylem parenchymal cells. Moreover, although root growth and xylem differentiation in antisense PAO tobacco (Nicotiana tabacum) plants were unaltered, overexpression of maize PAO (S-ZmPAO) as well as down-regulation of the gene encoding S-adenosyl-l-methionine decarboxylase via RNAi in tobacco plants promoted vascular cell differentiation and induced programmed cell death in root cap cells. Furthermore, following Spd treatment in maize and ZmPAO overexpression in tobacco, the in vivo H(2)O(2) production was enhanced in xylem tissues. Overall, our results suggest that, after Spd supply or PAO overexpression, H(2)O(2) derived from polyamine catabolism behaves as a signal for secondary wall deposition and for induction of developmental programmed cell death.     

Characterization of the maize xylem sap proteome

The xylem in plants has mainly been described as a conduit for water and minerals, but emerging evidence also indicates that the xylem contains protein. To study the proteins in xylem sap, we characterized the identity and composition of the maize xylem sap proteome. The composition of the xylem sap proteome in maize revealed proteins related to different phases of xylem differentiation including cell wall metabolism, secondary cell wall synthesis, and programmed cell death. Many proteins were found to be present as multiple isoforms and some of these isoforms are glycosylated. Proteins involved in defense mechanisms were also present in xylem sap and the sap proteins were shown to have antifungal activity in bioassays.     

Unravelling cell wall formation in the woody dicot stem

Populus is presented as a model system for the study of wood formation (xylogenesis). The formation of wood (secondary xylem) is an ordered developmental process involving cell division, cell expansion, secondary wall deposition, lignification and programmed cell death. Because wood is formed in a variable environment and subject to developmental control, xylem cells are produced that differ in size, shape, cell wall structure, texture and composition. Hormones mediate some of the variability observed and control the process of xylogenesis. High-resolution analysis of auxin distribution across cambial region tissues, combined with the analysis of transgenic plants with modified auxin distribution, suggests that auxin provides positional information for the exit of cells from the meristem and probably also for the duration of cell expansion. Poplar sequencing projects have provided access to genes involved in cell wall formation. Genes involved in the biosynthesis of the carbohydrate skeleton of the cell wall are briefly reviewed. Most progress has been made in characterizing pectin methyl esterases that modify pectins in the cambial region. Specific expression patterns have also been found for expansins, xyloglucan endotransglycosylases and cellulose synthases, pointing to their role in wood cell wall formation and modification. Finally, by studying transgenic plants modified in various steps of the monolignol biosynthetic pathway and by localizing the expression of various enzymes, new insight into the lignin biosynthesis in planta has been gained.