Developing xylem‐preferential expression of PdGA20ox1, a gibberellin 20‐oxidase 1 from Pinus densiflora,improves woody biomass production in a hybrid poplar

Woody biomass has gained popularity as an environmentally friendly, renewable and sustainable resource for liquid fuel production. Here, we demonstrate biotechnological improvement of the quantity and quality of woody biomass by employing developing xylem (DX)‐preferential production of gibberellin (GA), a phytohormone that positively regulates stem growth. First, for the proof of concept experiment, we produced transgenic Arabidopsis plants expressing GA20‐oxidase, a key enzyme in the production of bioactive GAs, from Pinus densiflora (PdGA20ox1) under the control of either a constitutive 35S promoter, designated 35S::PdGA20ox1, or a DX‐specific promoter (originated from poplar), designated DX15::PdGA20ox1. As we hypothesized, both transgenic Arabidopsis plants (35S::PdGA20ox1 and DX15::PdGA20ox1) exhibited an accelerated stem growth that resulted in a large increase of biomass, up to 300% compared to wild‐type control plants, together with increased secondary wall thickening and elongation of fibre cells. Next, we applied our concept to the production of transgenic poplar trees. Both transgenic poplar trees (35S::PdGA20ox1 and DX15::PdGA20ox1) showed dramatic increases in biomass, up to 300%, with accelerated stem growth and xylem differentiation. Cell wall monosaccharide composition analysis revealed that in both Arabidopsis and poplar, glucose and xylose contents were significantly increased. However, undesirable phenotypes of 35S::PdGA20ox1 poplar, including poor root growth and leaf development, were found. Interestingly, DX15::PdGA20ox1 poplar resulted in a reduction of undesirable phenotypes. Our results indicate that the controlled production of GAs through a tissue‐specific promoter can be utilized as an efficient biotechnological tool for producing enhanced plant biomass, minimizing unwanted effects.     

Stem growth,flower formation,and endogenous gibberellins in a normal and a dwarf strain of Silene armeria

The physiological basis of dwarfism in a single-gene, recessive mutant of Silene armeria L. was investigated through comparison with a normal strain. Exposure of the normal strain to long days led to stem growth and flower formation while similar exposure of the dwarf strain led only to flowering, with very little stem growth. Application of gibberellin A₃ or A₄₊₇ in short days promoted stem elongation in the normal strain, but had a much lesser effect in the dwarf strain. Upon extraction and chromatographic fractionation of the endogenous gibberellins (GAs) in the normal strain of S. armeria, three zones of GA activity were found. An increase in one zone of activity was found in both strains after 1 long day. Neither the quality nor the quantity of the extractable GAs differed greatly between the dwarf and the normal strain. Vegetative dwarf scions, grafted onto fully induced, normal stocks formed flowers, but their growth habit was not changed. Thus, the lack of stem growth in response to long days in the dwarf strain appears to result from a lack of GA sensitivity in the stem tissue of these plants. However, during flower formation dwarf plants did exhibit elongation of the peduncles. This response was suppressed by the growth retardant 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618), and applied GA₃ could partially overcome this inhibition. Thus, peduncle elongation in the dwarf strain appears to be regulated by endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - SD short day(s)     

Activation tagging of Arabidopsis POLYGALACTURONASE INVOLVED IN EXPANSION2 promotes hypocotyl elongation,leaf expansion,stem lignification,mechanical stiffening,and lodging

Pectin is the most abundant component of primary cell walls in eudicot plants. The modification and degradation of pectin affects multiple processes during plant development, including cell expansion, organ initiation, and cell separation. However, the extent to which pectin degradation by polygalacturonases affects stem development and secondary wall formation remains unclear. Using an activation tag screen, we identified a transgenic Arabidopsis thaliana line with longer etiolated hypocotyls, which overexpresses a gene encoding a polygalacturonase. We designated this gene as POLYGALACTURONASE INVOLVED IN EXPANSION2 (PGX2), and the corresponding activation tagged line as PGX2^AT. PGX2 is widely expressed in young seedlings and in roots, stems, leaves, flowers, and siliques of adult plants. PGX2‐GFP localizes to the cell wall, and PGX2^AT plants show higher total polygalacturonase activity and smaller pectin molecular masses than wild‐type controls, supporting a function for this protein in apoplastic pectin degradation. A heterologously expressed, truncated version of PGX2 also displays polygalacturonase activity in vitro. Like previously identified PGX1^AT plants, PGX2^AT plants have longer hypocotyls and larger rosette leaves, but they also uniquely display early flowering, earlier stem lignification, and lodging stems with enhanced mechanical stiffness that is possibly due to decreased stem thickness. Together, these results indicate that PGX2 both functions in cell expansion and influences secondary wall formation, providing a possible link between these two developmental processes.     

COMPARATIVE CYTOHISTOLOGICAL STUDIES ON INHIBITION AND PROMOTION OF STEM GROWTH IN CHRYSANTHEMUM MORIFOLIUM

Sachs , R. M. (U. California, Davis), and A. M. Kopranek . Comparative cytohistological studies on inhibition and promotion of stem growth in Chrysanthemum morifolium. Amer. Jour. Bot. 50(8): 772-779. Illus. 1963.—The present study with Amo, CCC, and Phosfon,³ 3 substances which inhibit stem elongation, shows that all inhibit subapical cell expansion and division in Chrysanthemum morifolium var. ‘Indianapolis Yellow.‘ Furthermore, GA,³ in preventing the inhibition of stem elongation, maintains subapical activity at normal or greater than normal levels. For comparative purposes concentrations of the retardants and GA have been selected which completely prevent or promote the maximum rate of stem elongation. Phosfon causes complete inhibition of root growth and almost completely prevents dry matter accumulation in the tops. However, GA does not prevent such deleterious effects. Thus, GA and the growth retardants are mutually antagonistic only with respect to stem elongation and not to other aspects of growth. Furthermore, none of the retardants inhibits transverse stem growth; on the contrary transverse cell expansion and division in the subapical tissues are stimulated by the retardants, and as a result the stems of such plants are thicker than normal. GA not only prevents the thickening effect of the retardants, but, at the doses applied, GA-treated stems are considerably thinner than those of the controls, having fewer and smaller cells across the pith, cortical, and vascular tissues. Apparently, then, there is a relationship between longitudinal and transverse growth in the subapical tissues such that if one is promoted, the other is inhibited.     

CONTROL OF FLOWER FORMATION BY GROWTH RETARDANTS AND GIBBERELLIN IN SAMOLUS PARVIFLORUS,A LONG-DAY PLANT

The growth retardants AMO–1618 and CCC inhibited flower formation and stem elongation in Samolus parviflorus, a long-day rosette plant, under inductive conditions. The vegetative growth of the plants, as measured by leaf formation, was affected only slightly, or not affected at all. Application of gibberellic acid (GA₃) reversed completely the inhibition both of flower formation and of stem elongation caused by AMO, but relatively larger amounts of GA were required to reverse the CCC inhibition of stem elongation than that of flower formation. When applied under short-day conditions, AMO had no effect on the level of applied GA required for flower induction. When applied following long-day treatment the retardant caused some reduction of flower formation after marginal numbers of long days, but had no effect when enough long days to cause 100% flower formation were given. Other evidence indicates that the growth retardants act by inhibiting the synthesis of endogenous gibberellin. In LD plants, at least part of the action of inductive environmental conditions consists in causing an increase of gibberellin synthesis, supporting the hypothesis that relatively high GA levels are necessary for the production of the floral stimulus in this group of plants, as in long-short-day plants. The experiments with CCC indicate that stem elongation and flower formation in Samolus can be separated, and that the effect of GA on flower formation is not necessarily dependent on its effect on stem elongation.     

The role of a class III gibberellin 2‐oxidase in tomato internode elongation

A network of environmental inputs and internal signaling controls plant growth, development and organ elongation. In particular, the growth‐promoting hormone gibberellin (GA) has been shown to play a significant role in organ elongation. The use of tomato as a model organism to study elongation presents an opportunity to study the genetic control of internode‐specific elongation in a eudicot species with a sympodial growth habit and substantial internodes that can and do respond to external stimuli. To investigate internode elongation, a mutant with an elongated hypocotyl and internodes but wild‐type petioles was identified through a forward genetic screen. In addition to stem‐specific elongation, this mutant, named tomato internode elongated ‐1 (tie‐1) is more sensitive to the GA biosynthetic inhibitor paclobutrazol and has altered levels of intermediate and bioactive GAs compared with wild‐type plants. The mutation responsible for the internode elongation phenotype was mapped to GA2oxidase 7, a class III GA 2‐oxidase in the GA biosynthetic pathway, through a bulked segregant analysis and bioinformatic pipeline, and confirmed by transgenic complementation. Furthermore, bacterially expressed recombinant TIE protein was shown to have bona fide GA 2‐oxidase activity. These results define a critical role for this gene in internode elongation and are significant because they further the understanding of the role of GA biosynthetic genes in organ‐specific elongation.     

Thermoinduction of genes encoding the enzymes of gibberellin biosynthesis and a putative negative regulator of gibberellin signal transduction in<Emphasis Type="Italic"> Eustoma grandiflorum</Emphasis>

Eustoma grandiflorum Shinn requires vernalization for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization, the expression levels of genes encoding enzymes of GA biosynthesis, copalyl diphosphate synthetase, GA 20-oxidase and GA 3-hydroxylase, were examined using two culitvars that show different responses to vernalization. The three genes were induced in a vernalization- and a cultivar-dependent manner. EgSPY, a putative negative regulator of GA signal transduction, was also induced during the vernalization period. The results suggest that the expression of the genes encoding GAs biosynthesis is regulated by vernalization. We postulate that EgSPY functions as a negative regulator of GA signal transduction during vernalization, inhibiting adventitious shoot elongation during vernalization.Communicated by K.K. Kamo     

Microbial methanol formation: A major end product of pectin metabolism

Various pectinolytic strains ofClostridium, Erwinia, andPseudomonas species produced methanol as a major end product during growth on pectin but not on glucose or polygalacturonic acid. Pectin metabolism ofClostridium butyricum strain 4P1 correlated with a final product concentration of 16 mM at the end of growth, and a 1:1 stoichiometry for methanol production and percent initial substrate methoxylation. Growth on pectin was associated with high activity of pectin methylesterase and the absence of methanol consumption. The ecological significance of pectin metabolism and the establishment of microbial methylotrophic metabolism in nature is discussed.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Gibberellins and the photoperiodic control of stem elongation in the long-day plant Agrostemma githago L.

Agrostemma githago is a long-day rosette plant in which transfer from short days (SD) to long days (LD) results in rapid stem elongation, following a lag phase of 7–8 d. Application of gibberellin A₂₀ (GA₂₀) stimulated stem elongation in plants under SD, while 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618, an inhibitor of GA biosynthesis) inhibited stem elongation in plants exposed to LD. This inhibition of stem elongation by AMO-1618 was overcome by simultaneous application of GA₂₀, indicating that GAs play a role in the photoperiodic control of stem elongation in this species. Endogenous GA-like substances were analyzed using reverse-phase high-performance liquid chromatography and the d-5 corn (Zea mays L.) assay. Three zones with GA-like activity were detected and designated, in order of decreasing polarity, as A, B, and C. A transient, 10-fold increase in the activity of zone B occurred after 8–10 LD, coincident with the transition from lag phase to the phase of rapid stem elongation. After 16 LD the activity in this zone had returned to a level similar to that under SD, even though the plants were elongating rapidly by this time. However, when AMO-1618 was applied to plants after 11 LD, there was a rapid reduction in the rate of stem elongation, indicating that continued GA biosynthesis was necessary following the transient increase in activity of zone B, if stem elongation was to continue under LD. It was concluded that control of stem elongation in A. githago involves more than a simple qualitative or quantitative change in the levels of endogenous GAs, and that photoperiodic induction alters both the sensitivity to GAs and the rate of turnover of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - LDP long-day plant(s) - SD short day(s)     

In vitro evaluation of Colombian plant extracts against Black Sigatoka (Mycosphaerella fijiensis Morelet)

In this work, 90 dichloromethane and methanol extracts obtained from 45 plants collected at the Natural Reserve Bremen – La Popa (Colombia) and at the Natural Regional Park Ucumarí (NRPU, Colombia) belonging to five botanical families were evaluated at 1000 mg/l, for their in vitro fungicide activity through the ascospore germ tube elongation and the measurement of the mycelial radial growth of Mycosphaerella fijiensis assays. The methanol extracts from the species Lycianthes acutifolia (Solanaceae) and Piper pesaresanum (Piperaceae); as well as, the dichloromethane extracts from P. pesaresanum and those from the Lauraceae family named Nectandra acutifolia and Ocoteca paulii, all inhibited M. fijiensis ascospore germination in 100% in the germinative tube elongation assay. With regards to the effects of the plant extracts on mycelial radial growth, the methanol extracts from P. pesaresanum and the dichloromethane one from N. acutifolia both showed 100% inhibition in this bioassay. Additionally, from the phytochemical screening on the dichloromethane and methanol extracts it was found that compounds such as alkaloids, phenols and terpenes were present in most of the extracts evaluated and they might be the cause of the antifungal activities reported.     

The role of gibberellin biosynthesis in the control of growth and flowering in Matthiola incana

Recently, it was found that stem elongation and flowering of stock Matthiola incana (L.) R. Br. are promoted by exogenous gibberellins (GAs), including GA₄, and also by acylcyclohexanedione inhibitors of GA biosynthesis, such as prohexadione‐calcium (PCa) and trinexapac‐ethyl (TNE). Here, because it was unclear how GA biosynthetic inhibitors could promote stem elongation and flowering, their effect on GA biosynthesis has been examined by quantifying endogenous GA levels; also, the sensitivity of stem elongation and flowering to various GAs in combination with the inhibitors was examined. Stem elongation and flowering were most effectively promoted by GA₄ when combined with PCa and, next in order, by 2,2‐dimethyl‐GA₄, PCa, GA₄+TNE, TNE, GA₉+PCa and by GA₄. There was little or no promotion by GA₁, GA₃, GA₉, GA₁₃, GA₂₀ and 3‐epi‐2,2‐dimethyl‐GA₄. Both the promotive effects of the acylcyclohexanediones on stem elongation and flowering, particularly when applied with GA₄, and the fact that TNE caused a build‐up of endogenous GA₄ imply that one effect of TNE at the lower dose involved an inhibition of 2β‐hydroxylation of GA₄ rather than an inhibition of 20‐oxidation and 3β‐hydroxylation of GAs which were precursors of GA₄. Overall, these results indicate that: (1) GAs with 3β‐OH and without 13‐OH groups (e.g. GA₄) are the most important for stem elongation and flowering in M. incana; (2) growth promotion rather than inhibition can result if an acylcyclohexanedione acts predominantly to slow 2β‐hydroxylation and so slows inactivation of active gibbberellins, including GA₄. It follows that a low dose of an acylcyclohexanedione can be a ‘growth enhancer’ for any applied GA that is liable to inactivation by 2β‐hydroxylation.     

Boron toxicity is alleviated by hydrogen sulfide in cucumber (Cucumis sativus L.) seedlings

Boron (B) is an essential micronutrient for plants, which when occurs in excess in the growth medium, becomes toxic to plants. Rapid inhibition of root elongation is one of the most distinct symptoms of B toxicity. Hydrogen sulfide (H₂S) is emerging as a potential messenger molecule involved in modulation of physiological processes in plants. In the present study, we investigated the role of H₂S in B toxicity in cucumber (Cucumis sativus) seedlings. Root elongation was significantly inhibited by exposure of cucumber seedlings to solutions containing 5 mM B. The inhibitory effect of B on root elongation was substantially alleviated by treatment with H₂S donor sodium hydrosulfide (NaHS). There was an increase in the activity of pectin methylesterase (PME) and up-regulated expression of genes encoding PME (CsPME) and expansin (CsExp) on exposure to high B concentration. The increase in PME activity and up-regulation of expression of CsPME and CsExp induced by high B concentration were markedly reduced in the presence of H₂S donor. There was a rapid increase in soluble B concentrations in roots on exposure to high concentration B solutions. Treatment with H₂S donor led to a transient reduction in soluble B concentration in roots such that no differences in soluble B concentrations in roots in the absence and presence of NaHS were found after 8 h exposure to the high concentration B solutions. These findings suggest that increases in activities of PME and expansin may underlie the inhibition of root elongation by toxic B, and that H₂S plays an ameliorative role in protection of plants from B toxicity by counteracting B-induced up-regulation of cell wall-associated proteins of PME and expansins.     

<Emphasis Type="Italic">CKB1</Emphasis> is involved in abscisic acid and gibberellic acid signaling to regulate stress responses in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Casein kinase II (CK2), an evolutionarily well-conserved Ser/Thr kinase, plays critical roles in all higher organisms including plants. CKB1 is a regulatory subunit beta of CK2. In this study, homozygous T-DNA mutants (ckb1-1 and ckb1-2) and over-expression plants (35S:CKB1-1, 35S:CKB1-2) of Arabidopsis thaliana were studied to understand the role of CKB1 in abiotic stress and gibberellic acid (GA) signaling. Histochemical staining showed that although CKB1 was expressed in all organs, it had a relatively higher expression in conducting tissues. The ckb1 mutants showed reduced sensitivity to abscisic acid (ABA) during seed germination and seedling growth. The increased stomatal aperture, leaf water loss and proline accumulation were observed in ckb1 mutants. In contrast, the ckb1 mutant had increased sensitivity to polyaluminum chloride during seed germination and hypocotyl elongation. We obtained opposite results in over-expression plants. The expression levels of a number of genes in the ABA and GA regulatory network had changed. This study demonstrates that CKB1 is an ABA signaling-related gene, which subsequently influences GA metabolism, and may play a positive role in ABA signaling.     

Average effect of a mutation in lignin biosynthesis in loblolly pine

Cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) is a monolignol biosynthetic enzyme that catalyzes the final step of lignin subunit biosynthesis in higher plants. Recently, a mutant allele of the cad gene, cad-n1, encoding for the CAD enzyme, was discovered in loblolly pine. By reducing the expression of the cad gene, this mutant has a decreased lignin content and major changes in the lignin composition in wood. In this study, we found that the substitution of a wild-type allele by cad-n1 was associated with a significant effect on 2nd-year shoot elongation in a half-sib family of loblolly pine (designated family 7–1037). The average effect of cad-n1 appeared to increase with tree growth and was greater for stem radial growth than height growth. An increase of 14.1% in de-barked volume in year 4 was associated with cad-n1. Co-segregation analysis indicated that the cad locus itself might represent a gene that governs stem growth in pine. The significance of the mutation cad-n1 for tree growth and wood processing is discussed. Received: 31 December 1998 / Accepted: 30 January 1999     

A mechanism coordinating root elongation,endodermal differentiation,redox homeostasis and stress response

Root growth relies on both cell division and cell elongation, which occur in the meristem and elongation zones, respectively. SCARECROW (SCR) and SHORT-ROOT (SHR) are GRAS family genes essential for root growth and radial patterning in the Arabidopsis root. Previous studies showed that SCR and SHR promote root growth by suppressing cytokinin response in the meristem, but there is evidence that SCR expressed beyond the meristem is also required for root growth. Here we report a previously unknown role for SCR in promoting cell elongation. Consistent with this, we found that the scr mutant accumulated a higher level of reactive oxygen species (ROS) in the elongation zone, which is probably due to decreased expression of peroxidase gene 3, which consumes hydrogen peroxide in a reaction leading to Casparian strip formation. When the oxidative stress response was blocked in the scr mutant by mutation in ABSCISIC ACID 2 (ABA2) or when the redox status was ameliorated by the upbeat 1 (upb1) mutant, the root became significantly longer, with longer cells and a larger and more mitotically active meristem. Remarkably, however, the stem cell and radial patterning defects in the double mutants still persisted. Since ROS and peroxidases are essential for endodermal differentiation, these results suggest that SCR plays a role in coordinating cell elongation, endodermal differentiation, redox homeostasis and oxidative stress response in the root. We also provide evidence that this role of SCR is independent of SHR, even though they function similarly in other aspects of root growth and development.