Gibberellic Acid Sensitivity Determines the Length of the Extension Zone in Wheat Leaves

To test the hypothesis that gibberellic acid (GA) sensitivityaffects the length of the extension zone (LEZ) of leaf No. 1of wheat seedlings, we performed a gene dosage experiment usingRht dwarfing genes that condition GA insensitivity. We utilizednearly isogenic lines, at Rht-dosage levels of 0, 2 and 4 alleles.Anatomical markers (distances between successive stomates) wereused to infer the distribution of growth along the axis of theleaf. Interstomatal distance (ISD) and LEZ were inverse linearfunctions of Rht-dosage. The number of stomates matured perhour was independent of Rht-dosage. The relationship betweenISD and distance along the axis within the extension zone (EZ)was indistinguishable from linear. Rht-dosage did not affectthe slope of the regression of ISD against distance along theEZ. A-REST (AR; ancymidol, a potent GA synthesis inhibitor)reduced LEZ. Wild type was more sensitive to AR than doubledwarf. AR affected growth of leaf No. 1 more than length ofthe coleoptile, regardless of Rht-dosage. AR-dosage affectedcell division, whereas Rht-dosage did not. Extension zone, elongation, gibberellic acid, Rht, wheat, Triticum aesiivum L.     

The effects of temperature and the Rht3 dwarfing gene on growth, cell extension, and gibberellin content and responsiveness in the wheat leaf

The effects of low temperature and the Rht3 dwarfing gene onthe dynamics of cell extension in leaf 2 of wheat were examinedin relation to gibberellin (GA) content and GA-responsivenessof the extension zone. Leaf 2 of wild-type (rht3) wheat closelyresembled that of the Rht3 dwarf mutant when seedlings weregrown at 10C. The maximum relative elemental growth rate (REGR)within the extension zone in both genotypes was lower at 10Cthan at 20C, but the position with respect to the leaf basewas unaffected by temperature. The size of the extension zoneand epidermal cell lengths were similar in both genotypes at10C. Growth at 20C, instead of 10C, increased the lengthof the extension zone beyond the point of maximum REGR in thewild type, but not in the Rht3 mutant. Increasing temperatureresulted in longer epidermal cells in the wild type. Treatingwild-type plants at 10C with gibberellic acid (GA₃) also increasedthe length of the extension zone, but the Rht3 mutant was GA-non-responsive.However, the concentrations of endogenous GA₁ and GA₃ remainedsimilar across the extension zone of wild-type plants grownat both temperatures, despite large differences in leaf growthrates. The period of accelerating REGR as cells enter the extensionzone, and the maximum REGR attained, are apparently not affectedby GA. It is proposed that GA functions as a stimulus for continuedcell extension by preventing cell maturation in the region beyondmaximum REGR and that low temperature increases the sensitivitythreshold for GA action. Key words: Cell extension, gibberellin, Rht3 dwarfing gene, temperature, wheat leaf     

The effect of gibberellic acid on the response of leaf extension to low temperature

The effect of cooling on leaf extension rate (LER) and on relative elemental growth rate (REGR) was measured in both gibberellic acid (GA)-responsive dwarf barley and in the same barley variety treated with GA. Seedlings were maintained at 20 degrees C while their leaf extension zone (LEZ) temperature was reduced either in steps to -6 degrees C in short-term cooling experiments, or to 10 degrees C for 48 h in long-term cooling experiments. Short-term cooling resulted in a biphasic response in LER, with a clear inflection point identified. Below this point, the activation energy for leaf extension becomes higher. The short-term response of LER to cooling was altered by the application of GA, which resulted in a lower base temperature (Tb), inflection point temperature and activation energy for leaf extension. Both GA-treated and untreated seedlings were less sensitive to cooling maintained for a prolonged period, with LER making a partial recover over the initial 5 h. Although long-term cooling reduced maximum REGR, it resulted in a longer LEZ and an increase in the length of mature interstomatal cells in GA-treated and untreated seedlings. These changes in overall physiology appear to enhance the ability of the leaves to continue expansion at suboptimal temperatures. In both GA-treated and cold-acclimated tissue, the occurrence of a longer LEZ was associated with a lower temperature sensitivity in LER.     

Effect of the Rht3 dwarfing gene on dynamics of cell extension in wheat leaves, and its modification by gibberellic acid and paclobutrazol

The second leaf of wheat was used as a model system to examinethe effects of the Rht3 dwarfing gene on leaf growth. Comparedto the rht3 wild type, the Rht3allele decreased final leaf length,surface area and dry mass by reducing the maximum growth rates,but without affecting growth duration. Gibberellic acid (GA₃)increased final leaf length and maximum growth rate in the rht3wild type, but was without effect on the Rht3 mutant, whichis generally regarded as being non-responsive to gibberellin(GA). Paclobutrazol, an inhibitor of GA biosynthesis, decreasedfinal leaf length and maximum growth rate in the rht3 wild typeto values similar to those in the untreated Rht3 mutant. NeitherGA₃ nor paclobutrazol affected the duration of leaf growth.The decrease in leaf length was produced by reduction of celllength rather than cell number. The maximum relative elementalgrowth rate (REGR) for cell extension was essentially the samein all treatments, as was the time between the cells leavingthe meristem and achieving maximum extension rate. The differencesbetween the genotypes and treatments were all almost entirelydue to differences in the time taken from the attainment ofmaximum REGR of cell extension to the cessation of extension.This was reflected in the length of the extension zone, whichwas approximately 6–8 per cent of final leaf length. Theeffects of the Rht3 allele, GA₃ and paclobutrazol all appearto be on the processes which promote the cessation of cell elongation. Key words: Cell extension, gibberellin, leaf growth, Rht3 gene, Triticum, wheat     

The Responsiveness to Temperature of the Extension Rates of Leaves of Wheat Growing in the Field under Different Levels of Nitrogen Fertilizer

The response of the rates of extension (LER) of wheat leaves(Triticum aestivum cv. Gamenya) to temperatures maintained fora short period was measured by changing the temperature of theextension zone and recording the changes in leaf length. Therange of temperatures used was from 4-38 °C. The LER ofall leaves responded to increases in temperature as field temperatureswere suboptimal. The data obtained from several series of measurementsover different ranges of temperature were combined to producea general response curve. The minimum temperature for LER wasconsidered to be approximately 0 °C, the optimum was 28.4°C, while the maximum was in excess of 38 °C. The responsivenessof LER to temperature, measured by the Q₁₀, declined exponentiallyfrom >6 at 5 °C to 2 at 20 °C. The Q₁₀ at 15 °Cwas not affected by nitrogen supply.     

Effects of NORIN 10 and Tom Thumb Dwarfing Genes on Morphology, Physiology and Abscisic Acid Production in Wheat

The pleiotropic effects of three genetically related dwarfinggenes were investigated in near-isogenic lines of wheat. TheNORIN 10 semi-dwarfing alleles, Rht 1 and Rht 2, and the TomThumb allele, Rht 3, were assessed for effects on some vegetativemorphological and physiological characters. The Rht allelesaffected leaf size with a resultant decrease in leaf area ofthe whole plant. Rht 3, which had the most marked effects, reducedleaf area in young plants by as much as 30 per cent. Althoughflag leaf dimensions and stomatal distributions of the flagleaf were altered, the gene had no effect on its area, stomatalconductance or net CO₂ exchange rate. Comparisons of Rht andtall plants revealed no differences in the abscisic acid (ABA)levels of either turgid or partially dehydrated leaves. Triticum aestivum L., wheat, dwarfing genes, leaf structure, abscisic acid, stomatal conductance, CO₂, exchange, relative growth rate     

The Effects of Dwarfing Genes Rht1 and Rht2 on Cellular Dimensions and Rate of Leaf Elongation in Wheat

The gibberellin insensitivity genes, Rht1 and Rht2, reducedepidermal cell lengths in leaves of isogenic lines of field-and laboratory-grown wheat (Triticum aestivum L.). Rht dosagesof zero (wild type), two (semi-dwarf) and four alleles (doubledwarf) had a linear negative effect on cell length in flag leavesof field-grown plants, and in the sheaths and blades of leafnumber 1 in laboratory grown plants. Decrease in cell length,rather than reduced cell number, accounted for most to all ofthe reduction in blade and sheath length. In sheaths, cell widthincreased with Rht dosage, but not sufficiently to compensatefor decreased length in determining average projected surfacearea. Rates of extension of leaf number 1 in laboratory-grownplants were negatively and linearly correlated with Rht dosage.Maximal growth rate was maintained longer in wild type thanin double dwarf, but the total duration of measurable extensionin leaf number 1 was not affected by Rht dosage. Cell size, elongation, Rht, wheat, Triticum aestivum L     

The Net Orientation of Wall Microfibrils in the Outer Periclinal Epidermal Walls of Seedling Leaves of Wheat

Use of the dichroic stain chlor-zinc-iodine revealed that thenet orientation of cellulose wall microfibrils in the outerparadermal wall of the epidermis of seedling wheat leaves isprincipally transverse in the extension zone. The net orientationof microfibrils changes abruptly to principally longitudinalat the end of cell elongation. The net angle of orientationof microfibrils in the extension zone was not a function ofRht-dosage (number of dwarfing alleles), and neither leaf extensionrate nor estimated maximum relative elemental rate of elongationwere functions of microfibril orientation. The results indicatethat elongation rates are not regulated by the net angle oforientation of microfibrils and support the concept that leafextension rate is regulated by the length of the extension zone.Copyright1995, 1999 Academic Press Cellulose wall microfibrils, extension zone, elongation, Rht, wheat, Triticum aestivum L     

Grass Leaf Elongation Rate as a Function of Developmental Stage and Temperature: Morphological Analysis and Modelling

Elongation of successive leaves was measured following defoliationof tall fescue plants in controlled environments. Measurementswere made under constant temperatures of 24 °C and 14 °C,and after temperature changes from 24 to 14 °C andvice versa.A morphological analysis of the growing leaf was made from thetime it was 1 mm long until it was fully elongated. The timeelapsed from initiation until the leaf was 1 mm long was estimated.Young leaves less than 1.5 mm long elongated slowly at a constantleaf elongation rate (LER). By extrapolating this LER back toleaf initiation from the apex it was calculated that elongationlasted 42.5 d at 24 °C and 51 d at 14 °C. Lengths ofthe division zone (DZ) and the extension-only zone (E-OZ) increasedto a maximum and then decreased during leaf development. Temperaturechange had an immediate effect on LER but the response varieddepending on the direction of the temperature change. To describethese different features, an empirical model of DZ and E-OZwas designed. Its five parameters were optimized at constanttemperature. The model was then used to simulate the LER ofplants subjected to temperature changes. Instant and lastingeffects of the initial temperature on mean LER in plants transferredfrom 14 to 24 °C andvice versawere well simulated. It wasconcluded that the major reason for differences was due to thegrowth stage (DZ and E-OZ lengths) at which the changes occurredat both temperatures.Copyright 1999 Annals of Botany Company Festuca arundinaceaSchreb., tall fescue, growth zone, division zone.     

Effect of Temperature on Gibberellin (GA) Responsiveness and on Endogenous GA(1) Content of Tall and Dwarf Wheat Genotypes

Near-isogenic wheat (Triticum aestivum L.) lines differing in height-reducing (Rht) alleles were used to investigate the effects of temperature on endogenous gibberellin (GA) levels and seedling growth response to applied GA₃. Sheath and lamina lengths of the first leaf were measured in GA treated and control seedlings, grown at 11, 18, and 25°C, of six Rht genotypes in each of two varietal backgrounds, cv Maris Huntsman and cv April Bearded. Endogenous GA₁ levels in the leaf extension zone of untreated seedlings were determined by gas chromatography-mass spectrometry with a deuterated internal standard in the six Maris Huntsman Rht lines grown at 10 and 25°C. Higher temperature increased leaf length considerably in the tall genotype, less so in the Rht1 and Rht2 genotypes, and had no consistent effect on the Rht1+2, Rht3 and Rht2+3 genotypes. In all genotypes, endogenous GA₁ was higher at 25°C than at 10°C. At 10°C the endogenous GA₁ was at a similar level in all the genotypes (except Rht2+3). At 25°C it increased 1.6-fold in the tall genotype, 3-fold in Rht1 and Rht2, 6-fold in Rht3, and 9-fold in Rht1+2. Likewise, the genotypic differences in leaf length were very conspicuous at 25°C, but were only slight and often unsignificant at 11°C. The response of leaf length to applied GA₃ in the Rht1, Rht2, and Rht1+2 genotypes increased significantly with lowering of temperature. These results suggest the possibility that the temperature effect on leaf elongation is mediated through its effect on the level of endogenous GA₁ and that leaf elongation response to endogenous or applied GAs is restricted by the upper limits set by the different Rht alleles.     

In vitro expression of genes affecting whole plant phenotype — the effect of Rht/Gai alleles on the callus culture response of wheat (Triticum aestivum L. em. Thell)

Summary Calli were initiated from immature embryos of 12 lines of hexaploid wheat (Triticum aestivum L. em. Thell). The lines were from 3 varieties — April Bearded, Bersee and Maris Huntsman — isogenic for the reduced height/gibberellic acid insensitivity (Rht) genes — Rht1, Rht2 and Rht3 — and the tall (rht) allele. The dwarfing genes had significant effects on the growth and morphogenesis of calli. The genes interacted with the 2,4-D in the medium and the varietal background. Calli of each line were cultured in the presence and absence of 1 mg/l of gibberellic acid (GA), but there was no interaction of the Rht genes with GA in vitro. The effect of the Rht genes is discussed in relation to their effects on cellular hormone metabolism and their involvement in previously described chromosome 4B effects in culture.     

Influence of Gibberellic Acid and the Rht3 Gene on Choline and Phospholipid Metabolism in Wheat Aleurone Tissue

The effect of gibberellic acid (GA3) on phospholipid metabolismand -amylase production was studied in aleurone tissue of twonear-isogenic lines of wheat (Triticum aesuvum L.). Incubationof embryoectomized seeds from a GA-responsive line (rht3, tall)with GA₃ caused the induction of -amylase activity after a lagphase of 30 h. In the case of embryoectomized seeds from a ‘GA-insensitive’line (Rh13, dwarf), however, the lag phase was extended up to50 h. During the first 14 h following imbibition, GA₃ inhibitedcholine uptake and its subsequent incorporation into phosphatidylcholine in the Rhr3 line but not in the rht3 line. GA₃ promotedphospholipid breakdown in both the lines during this period,however. GA₃ also terminated independent turnover of the cholineN-methyl groups in phosphatidyl choline and promoted turnoverof the whole choline headgroup. These results are discussedin relation to the possibility that phosphatidyl choline turnoveris an integral part of the GA₃ signal-transduction mechanismin aleurone tissue. Key words: GA3, Rht3 gene, choline, phospholipid     

Measuring the RGR of Individual Grass Plants

Vegetative growth of grasses was analysed by dry mass increaseof growing leaves.Holcus lanatuswas grown in a controlled environmentand leaf extension rates of leaf numbers 5–10 of the maintiller were monitored daily. Leaf appearance and leaf extensionrates (LER) of leaves 5–7 enabled the prediction of thefinal length and dry mass of leaf 8 during its growth. A linearincrease of leaf mass per unit leaf length (LML) of leaf 8 wasobserved during growth. After harvest the daily increase indry mass of growing leaves was calculated from the LER and correspondingincrease in LML. The relative growth rate (RGR) of the maintiller showed day-to-day fluctuations and was gradually reducedby 50% over a 16-d period. The RGR of the shoot was maintainedby tillering. The RGR of a single (grass) plant can be calculatedfrom four parameters only: LER, LML, leaf appearance and tillering.Variation of RGR over a period can be reconstructed after harvestand the impact of these four parameters on RGR can be established.Copyright1998 Annals of Botany Company. Relative growth rate, grass, leaf growth,Holcus lanatus.     

Gibberellin-induced changes in growth anisotropy precede gibberellin-dependent changes in cortical microtubule orientation in developing epidermal cells of barley leaves. Kinematic and cytological studies on a gibberellin-responsive dwarf mutant, M489

We conducted kinematic and cytological studies on "between vein" epidermal cells of the gibberellin (GA)-deficient M489 dwarf mutant of barley (Hordeum vulgare L. Himalaya). GAs affect radial and axial components of cell expansion and cortical microtubule orientation. Adaxial cells in particular expand radially after leaving the elongation zone (EZ), probably as part of leaf unrolling. Exogenous gibberellic acid corrects the mutant's short, wide blades, short EZ, and slow elongation rate. Cell production rates increase more on the adaxial than on the abaxial surface. Cells spend equal periods of time elongating in dwarf and tall plants, but relative elemental growth rates start to decline sooner in the dwarf. GA increased the rate at which longitudinal wall area increased because the increased axial growth more than compensated for reduced radial growth. In dwarf leaves, increased radial expansion was detected in basal parts of the EZ before cortical microtubules lost transverse orientation in the distal elongation zone. We conclude that loss of microtubule orientation is not required for low GA levels to reduce growth anisotropy.     

The Effects of Gibberellins on the Growth of Excised Tomato Roots

1. At appropriate concentrations both gibberellic acid (GA) and1-naphthalene-acetic acid (NAA) enhance the main axis growthof excised tomato roots grown in culture media containing sucroseat concentrations below 1 per cent. Lateral root extension growthis enhanced by GA at all sucrose concentrations tested; onlyat the lower sucrose concentrations is this effect observedwith NAA. Both GA and NAA increase the number of emergent lateralroots and this effect is most marked in media of low sucrosecontent. Both GA and NAA at higher concentrations inhibit rootgrowth but NAA exhibits its full range of growth effects overa much narrower concentration range than GA.
2. GA, like NAA,speeds up the loss of meristematic activity whichoccurs whenindividual meristems are repeatedly subculturedin media containing1 per cent, or higher concentrations ofsucrose.
3. The promotionof main axis growth by both GA and NAA involvesenhanced cellelongation and cell division. At a moderatelyinhibitory concentrationGA reduces both cell elongation andcell division; this is notthe case with NAA.
4. Gibberellins A₁, A₂, and A₄ resemble GA(gibberellin A₃) intheir growth effects. Allogibberic acidlike G A promotes lateralroot extension growth but causes markedinhibition of root growthat a much lower concentration thanGA.

     

Leaf Extension Rate in Temperate Pasture Grasses in Relation to Assimilate Pool in the Extension Zone

In two growth cabinet experiments the leaf extension rate (LER)was studied under a 14 h photoperiod followed by prolonged darkness,in tillers of the perennial temperate pasture grasses Phalaristuberosa L. cv. Sirosa and Dactylis glomerata L. cv. Currie.Levels of soluble non-structural carbohydrates and total     

A Model of the Leaf Extension Rate of Tall Fescue in Response to Nitrogen and Temperature

The leaf extension rate (LER) of tall fescue (Festuca arundinaceaSchreb.) was studied in the field under various nitrogen andtemperature regimes. The LER was closely related to temperaturewhen N was not limiting plant growth. Two distinct relationshipsbetween the LER and the temperature were obtained, one for vegetativegrowth and one for the reproductive period. These relationships,described by a Gompertz function, were exponential at temperaturesbelow 8 °C and linear at temperatures above 8 °C. Theymade possible the calculation of an optimal LER correspondingto non-limiting N conditions for plant growth. The strong influence of the temperature on the LER was stillobserved under N limiting conditions. The N status of the swardswas described by the ratio between the actual N content (N_actual)and the optimal N content (N_optimal). The N_optimal was definedas the N content experienced at a non-limiting level of N nutritionbut without N luxury consumption. The N_optimal, expressed asa function of dry matter yield, declined during growth. Theeffect of the N status of the swards on the LER was analysedby calculating the ratio between the actual LER and the optimalLER, and relating it to the ratio between N_actual and N_optimal.It was shown that these two ratios were highly correlated. Leaf extension, Festuca arundinacea, nitrogen, temperature     

Gibberellins and leaf expansion in near-isogenic wheat lines containing Rht1 and Rht3 dwarfing alleles

In near-isogenic lines of winter wheat (Triticum aestivum L. cv. Maris Huntsman) grown at 20° C under long days the reduced-height genes, Rht1 (semi-dwarf) and Rht3 (dwarf) reduced the rate of extension of leaf 2 by 12% and 52%, respectively, compared with corresponding rht (tall) lines. Lowering the growing temperature from 20° to 10° C reduced the rate of linear extension of leaf 2 by 2.5-fold (60% reduction) in the rht3 line but by only 1.6-fold (36% reduction) in the Rht3 line. For both genotypes, the duration of leaf expansion was greater at the lower temperature so that final leaf length was reduced by only 35% in the rht3 line and was similar in the Rht3 line at both temperatures. Seedlings of the rht3 (tall) line growing at 20° C responded positively to root-applied gibberellin A₁ (GA₁) in the range 1–10 μM GA₁; there was a linear increase in sheath length of leaf 1 whereas the Rht3 (dwarf) line remained unresponsive. Gibberellins A_{1, 3, 4, 8, 19, 20, 29, 34, 44} and ₅₃ were identified by full-scan gas chromatography-mass spectrometry in aseptically grown 4-d-old shoots of the Rht3 line. In 12-d-old seedlings grown at 20° C, there were fourfold and 24-fold increases in the concentration of GA₁ in the leaf expansion zone of Rht1 and Rht3 lines, respectively, compared with corresponding rht lines. Although GA₃ was present at a similar level to GA₁ in the rht3 (tall) line it accumulated only fivefold in the Rht3 (dwarf) line. The steady-state pool sizes of endogenous GAs were GA₁₉ ? GA₂₀ = GA₁ in the GA-responsive rht3 line whereas in the GA non-responsive Rht3 line the content of GA₁₉≈ GA₂₀ ? GA₁. It is proposed that one of the consequences of GA₁ action is suppression of GA₁₉-oxidase activity such that the conversion of GA₁₉ to GA₂₀ becomes a rate-limiting step on the pathway to GA₁ in GA-responsive lines. In the GA-non-responsive Rht lines it is suggested that GA₁₉ oxidase is not downregulated to the same extent and GA₁ accumulates before the next rate-limiting step on the pathway, its 2β-hydroxylation to GA₈. The steady-state pool sizes of GA_{19, 20, 1, 3} and ₈ were similar in developmentally equivalent tissues of the rht3 (tall) line growing at 10° C and 20° C despite a 2.5-fold difference in the rate of leaf expansion. In contrast, in the Rht3 (dwarf) line, the extent of accumulation of GA₁ reflected the severity of the phenotype at the two temperatures with slower growing tissues accumulating less, not more, GA₁. These results are interpreted as supporting the proposed model of regulation of the GA-biosynthetic pathway rather than previous suggestions that GA₁ accumulates in GA-insensitive dwarfs as a consequence of reduced growth rates.