Coordinated action of β‐galactosidases in the cell wall of embryonic axes during chickpea germination and seedling growth

The plant cell wall is a dynamic structure whose constant modification is necessary for plant cells to grow and divide. In the cell walls of chickpea (Cicer arietinum) there are at least four β‐galactosidases, whose presence and location in embryonic axes during the first 48 h of seed imbibition are discussed in this paper. We examined their roles as cell wall‐modifying enzymes in germinative and/or post‐germinative events. At the start of germination, only βV‐Gal, and to a lesser extent βIV‐Gal, appear in the axes before rupture of the testa, suggesting they are related to germination sensu stricto. Once the testa has broken, the four β‐galactosidases are involved in growth and differentiation of the axes. Immunolocation of the different proteins in axes, which in part confirms previous results in seedlings and plants, allows assignment of post‐germinative roles to βI‐Gal and βIII‐Gal as cell wall modifiers in vascular tissue elements. βIV‐Gal and βV‐Gal participate in the initial events of germination in which cell walls are involved: βV‐Gal in cell proliferation, detachment of root cap cells and initial vascular tissue differentiation; both of them in xylem maturation; and βIV‐Gal in thickening of the primary cell wall. Together with other cell wall‐modifying enzymes, such as expansins and XTH, chickpea galactosidases might function in a sequential order in turnover of the primary cell wall, allowing the elongation of embryonic axes during seed germination.     

Increased expression of two cDNAs encoding metallothionein‐like proteins during growth of Cicer arietinum epicotyls

The present study was undertaken to identify and characterize clones whose expression increase during Cicer arietinum epicotyl growth. Two cDNAs encoding two different plant metallothionein (MT)‐like proteins have been isolated from a cDNA library from epicotyls of Cicer arietinum L. cv. Castellana. The CanMT‐1 deduced protein appears to have the typical structure of type 1 MT where all Cys residues are in Cys‐X‐Cys motifs, while the CanMT‐2 has the typical structure of type 2 MT having Cys‐Cys and Cys‐X‐X‐Cys motifs within the N‐terminal domain. Both chickpea CanMTs are up‐regulated during epicotyl growth, showing increased expression in mature tissues, mostly CanMT‐1, which is undetectable in young epicotyls. Accordingly, stem of chickpea plants displayed the highest level of CanMT‐1 expression in the basal internode, with reduced growth, decreasing towards the apex. Osmotic stress by PEG, which inhibited growth, and ABA treatment induced the expression of MT‐like genes, which points to a relationship between chickpea MTs and ABA‐mediated stress response. Unlike CanMT‐2, CanMT‐1 is induced in chickpea epicotyls by cadmium indicating a different function for both clones. We conclude that these MT‐like proteins, in particular CanMT‐1, are regulated by the developmental stage and may participate in cell maturation process.     

Hypergravity increases the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity in azuki bean epicotyls.

Elongation growth of dark-grown azuki bean (Vigna angularis Ohwi et Ohashi cv. Takara) epicotyls was suppressed by hypergravity at 30 x g and above. Acceleration at 300 x g significantly decreased the mechanical extensibility of cell walls. The amounts of cell wall polysaccharides (pectin, hemicellulose-II and cellulose) per unit length of epicotyls increased under the hypergravity condition. Hypergravity also increased the amounts and the weight-average molecular mass of xyloglucans in the hemicellulose-II fraction, while decreasing the activity of xyloglucan-degrading enzymes extracted from epicotyl cell walls. These results suggest that hypergravity increases the amounts and the molecular mass of xyloglucans by decreasing xyloglucan-degrading activity. Modification of xyloglucan metabolism as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of azuki bean epicotyls.     

The immunolocation of a xyloglucan endotransglucosylase/hydrolase specific to elongating tissues in Cicer arietinum suggests a role in the elongation of vascular cells

In a previous work, a Cicer arietinum cDNA clone (CaXTH1) encoding a xyloglucan endotransglucosylase/hydrolase (XTH1) protein was isolated and characterized. CaXTH1 showed an expression pattern specific to growing tissue: mostly epicotyls and the upper growing internodes of adult stems. CaXTH1 mRNA was not detected in any other organs of either seedlings or adult plants, suggesting an involvement of the putative XTH encoded by CaXTH1 in the chickpea cell expansion process. After the generation of polyclonal antibodies by using the XTH1 recombinant protein and the analysis of the specificity of the antibodies for XTH proteins, here the specific location of the chickpea XTH1-cross-reacting protein in cell walls of epicotyls, radicles, and stems is reported, evaluated by western blot and immunocytochemical studies. The results indicate a function for this protein in the elongation of parenchyma cells of epicotyls and also in developing vascular tissue, suggesting a role in the elongation of vascular cells.     

Xyloglucan oligosaccharides cause cell wall loosening by enhancing xyloglucan endotransglucosylase/hydrolase activity in azuki bean epicotyls

Addition of xyloglucan-derived oligosaccharides shifted the wall-bound xyloglucans to a lower molecular mass distribution and increased the cell wall extensibility of the native epidermal tissue strips isolated from azuki bean (Vigna angularis) epicotyls. To ascertain the mechanism of oligosaccharide function, we examined the action of a xyloglucan endotransglucosylase/hydrolase (XTH) showing both endotransglucosylase and endohydrolase activities, isolated from azuki bean epicotyl cell walls, in the presence of xyloglucan oligosaccharides. The addition of xyloglucan oligosaccharides enhanced the xyloglucan-degrading activity of XTH against isolated xyloglucan substrates. When the methanol-fixed epidermal tissue strips were incubated with XTH, the molecular mass of wall-bound xyloglucans was decreased and the cell wall extensibility increased markedly in the presence of the oligosaccharides. These results suggest that xyloglucan oligosaccharides stimulate the degradation of xyloglucans by enhancing the XTH activity within the cell wall architecture, thereby increasing the cell wall extensibility in azuki bean epicotyls.     

Graviperception in growth inhibition of plant shoots under hypergravity conditions produced by centrifugation is independent of that in gravitropism and may involve mechanoreceptors

Hypergravity caused by centrifugation inhibits elongation growth of shoots by decreasing the cell wall extensibility via suppression of xyloglucan breakdown as well as by the thickening of cell walls. The mechanism of graviperception in hypergravity-induced growth inhibition was investigated in Arabidopsis [A. thaliana (L.) Heynh.] hypocotyls and azuki bean (Vigna angularis Ohwi et Ohashi) epicotyls. Hypergravity caused growth suppression in both sgr1-1 and pgm1, which are Arabidopsis mutants deprived of gravitropism, as in wild-type plants, suggesting that the graviperception in hypergravity-induced growth inhibition of shoots is independent of that in gravitropism. Hypergravity had no effects on growth of azuki bean epicotyls or Arabidopsis hypocotyls in the presence of lanthanum or gadolinium, which are blockers of mechanoreceptors. Moreover, lanthanum or gadolinium at the same concentration had no influence on gravitropism of azuki bean epicotyls and Arabidopsis hypocotyls. Hypergravity had no effects on cell wall extensibility and affected neither xyloglucan metabolism nor the thickness of cell walls in the lanthanum- or gadolinium-treated azuki bean epicotyls. Lanthanum or gadolinium inhibited the hypergravity-induced increase in the pH of the apoplastic fluid in the epicotyls, which is involved in the processes of the suppression of xyloglucan breakdown due to hypergravity. These findings suggest that plants perceive the hypergravity stimuli by mechanoreceptors in the plasma membrane, and utilize the perceived signal to regulate the growth rate of their shoots.Abbreviations HC-I Hemicellulose-I - HC-II Hemicellulose-II     

Role of xyloglucan in gravitropic bending of azuki bean epicotyl

The mechanism of the gravitropic bending was studied in azuki bean epicotyls. The cell wall extensibility of the lower side became higher than that of the upper side in the epicotyl bending upward. The contents of matrix polysaccharides of the cell wall (pectin and xyloglucan in hemicellulose-II) in the lower side became smaller than those in the upper side. The molecular mass of xyloglucans in the lower side decreased. After an epicotyl was fixed to a metal rod to prevent the bending, gravistimulation was applied. Fundamentally the same results were obtained with respect to rheological and chemical characteristics of the cell wall as those of epicotyls showing gravitropic bending. The present results suggested that the initial gravitropic bending was caused by the increase in extensibility of the lower side and the decrease in extensibility of the upper side via the change of the cell wall matrix, especially xyloglucans.     

Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls

Plant cell walls contain a glycoprotein component rich in the otherwise rare amino acid hydroxyproline. We examined the synthesis and accumulation of wall hydroxyproline during different states of elongation growth in pea epicotyls. Light-grown peas contained more wall hydroxyproline than their taller, dark-grown counterparts. When elongation was studied by marking growing stems in situ, there was a marked accumulation of wall hydroxyproline coincident with the cessation of elongation. Dividing and elongating regions of the epicotyl showed less wall hydroxyproline than did regions where elongation was no longer occurring.Hydroxyproline biosynthesis was examined by incubation of excised sections of tissues in various growth states in ¹⁴C-proline. The extent of conversion of these residues to ¹⁴C-hydroxyproline served as a measure of the rate of hydroxyproline synthesis. This rate was highest in tissues which had ceased elongation. The low rate of hydroxyproline synthesis in dividing and elongating cells was probably not due to the inability to hydroxylate peptidyl proline or to secrete proteins.These data show a positive correlation between the synthesis and accumulation of cell wall hydroxyproline and the cessation of cell elongation in pea epicotyls.     

Xyloglucan antibodies inhibit auxin-induced elongation and cell wall loosening of azuki bean epicotyls but not of oat coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl₁Glc₁), xyloglucan heptasaccharides (Xyl₃Glc₄), and octasaccharides (Gal₁Xyl₃Glc₄). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.     

Endomitosis and the effect of gibberellic acid in different Pisum sativum L. cultivars

The evolution of the total amount of DNA in epicotyls and of the amount of DNA per cell nucleus in epicotyl cortex cells during germination was followed in two closely related pea varieties, Pisum sativum cv. Finale and Pisum sativum cv. Rondo. Under etiolating conditions, growth of the cv. Rondo occurs only by cell elongation which is preceded by endomitotic DNA synthesis, while in the cv. Finale growth is the result of cell elongation accompanied by endomitotic DNA synthesis and cell division. The maximum C-level attained in both cultivars under etiolating conditions is 8 C (C=haploid amount of DNA in a gamete cell). Both the maximum C-level reached and the percentage of cells reaching this C-level seem to be under strict genetic control. In both cultivars, light inhibits the endomitotic DNA replication.Neither gibberellic acid (GA₃), nor AMO 1618 alter the maximum C-level or the percentage distribution of the C-classes. Both growth regulators are effective, although in an opposite way, only in tissues where cell division occurs or where endomitotic DNA synthesis is blocked, as in light-grown pea epicotyls.