Internode length in Pisum. Two further gibberellin-insensitivity genes, lka and lkb

Two further internode length genes are identified in Pisum sativum L. and named lka (identified from line NGB5865) and lkb (from NGB5862). These genes result in a similar phenotype, which includes reduced stem elongation, peduncle length and basal branching, and 'banding' of the stem. These effects are similar to, but less severe than, those of gene lk . Genes lka and lkb influence gibberellin (GA) sensitivity, since mutants NGB5865 and NGB5862 possess similar levels of endogenous GA-like substances to the wild-type parental cultivar Torsdag and respond less to applied GA₁ than do wild-type plants or GA-synthesis mutants of a similar stature. The action of genes lka and lkb is localised in the young apical tissue but is not thought to involve GA-perception, since plants possessing genes lka and lkb are not true phenocopies of GA-deficient plants. The genetic interaction of genes lka and lkb is examined and the action of gene lkb on a le gene background determined.     

Internode length in Pisum. Estimation of GA1 levels in genotypes Le, le and led

The levels of GA₁, 3-epiGA₁ and GA₈ in genotypes Le, le and le^d of Pisum sativum L. were determined by gas chromatography-selected ion monitoring (GC-SIM) after feeds of [³H, ¹³C]-GA₂₀ to each genotype. The levels of endogenous and [¹³C]-labelled metabolites were determined by reverse isotope dilution with unlabelled GA₁, 3-epiGA₁ and GA₈. The results demonstrate a quantitative relationship between the level of GA₁ and the extent of elongation both on a per plant and a per g fresh weight basis. These results are consistent with previous findings in peas and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis.
The levels of 3-epiGA₁ also decreased in the genotypic sequence Le, le, le^d although not as rapidly as for the level of GA₁. This may suggest that the alleles at the le locus also influence the formation of 3-epiGA₁.     

Internode length in Pisum. A new allele at the Lh locus

A new allele at the Lh locus has been identified in Pisum sativum L. and named lhⁱ . This allele results in reduced GA levels in young shoots, and a dwarf phenotype. Gas chromatography-selected ion monitoring (GC-SIM) with dideuterated internal standards has been used to demonstrate a quantitative relationship between the level of endogenous GA₁ and internode length using the three alleles ( Lh, lh and lhⁱ ) at the Lh locus. These results are consistent with previous findings in peas (for alleles at the Le locus) and other species possessing a predominant early 13-hydroxylation pathway for GA biosynthesis and support the role of GA₁ as the major native GA in peas with biological activity in its own right. However, in contrast to alleles at the Le locus, GA₂₀ levels are also reduced in lh and lhⁱ plants. The lhⁱ allele also has possible pleiotropic effects on seed abortion, leading to a reduction in seed yield compared to plants homozygous for the previously characterised Lh or lh alleles.     

Internode length in Pisum. Action of gene lw

Jolly, C. J., Reid, J. B. and Ross, J. J. 1987. Internode length in Pisum. Action of gene lw.
Mutant K29 of Pisum sativum L. is shown to possess a recessive gene at a new locus, lw , which results in reduced internode length, delayed flowering and increased symptoms of water congestion compared with the parental cv. Torsdag. The interaction of gene lw with the internode length genes na, le, la and cry ⁵ is examined. Extracts from the shoots of Iw plants are shown to contain similar levels of gibberellin (GA)-like substances to comparable Lw plants, but Iw plants do not elongate to the same extent as Lw plants when treated with GA₁₉ GA₁₉, or GA₂₀. The effect of gene Iw is not graft-transmissible. Unlike essentially isogenic dwarf lines possessing the GA-synthesis genes le, Ih or Is, lw plants show a relative increase in elongation similar to Torsdag in response to photoperiod extensions from sources rich in far-red light. These results suggest that gene lw probably does not reduce elongation by influencing GA-synthesis and that the response to photoperiod extensions with far-red light may depend on the level of GA.     

Internode length in Pisum. Comparison of genotypes in the light and dark

There is a strong relationship across the full range of gibberellin deficient, internode length genotypes ( le, lh, is, na ) between internode length in the dark and in red or white light. Further, the new, more severe allele at the le locus. Ie ^d, is shown to influence growth in the dark as well as in the light. These results suggest that darkeness does not specifically overcome any of the steps blocked by the gibberellin (GA) synthesis genes contrasting with the conclusions drawn by other workers. Supporting this conclusion in relation to the Ie gene are results which show that, at least at certain dosage rates, dark-grown Ie na plants respond better to GA₁ than to GA₂₀ similar to the response previously reported in light grown plants.
The greater response by plants of the nana line NGB1766 ( na ) to GA₁ in the dark than in the light suggests that light may influence internode length by altering GA-sensitivity. These results are discussed in relation to previous views on the control of stem elongation by light.     

Internode length in Pisum. Further studies on the 'micro' gene, lm

Reid, J. B. and Ross, J. J. 1988. Internode length in Pisum. Further studies on the 'micro' gene, lm . - Physiol. Plant. 72: 547–554.
In the garden pea, Pisum sativum L., gene lm confers the micro phenotype. The shoots of lm plants may be described as scaled-down versions of comparable Lm plants, with reduced internode length, leaflet size and rate of leaf expansion. However, the first phenotypic effect of gene lm is on root morphology. The gene results in curling and reduced elongation of the roots and, eventually, degeneration of the root cortex. These changes commence prior to any major visible effects in the shoot. The primary action of the lm gene does not appear to be confined to the root system, however, since epicotyl grafts between Lm and lm plants showed no graft-transmissible effects. The effects of gene lm are also apparent in dark-grown plants. Microdwarf plants ( lm le ) respond well to gibberellin A₁ (GA₁), but do not elongate to the same extent as dwarf ( Lm le ) plants. The two genotypes contain the same complement of GA-like substances. It is argued that gene lm is unlikely to be directly involved with GA-metabolism or the reception of the GA signal, but rather reduces the GA response by influencing some aspect of normal cell development, which results in the wide range of pleiotropic effects observed. Consequently, it may be misleading to continue to classify this gene simply as an internode length mutant.     

Internode length in Pisum. The involvement of ethylene with the gibberellin-insensitive erectoides phenotype

The gene lk in peas ( Pisum sativum L.) confers the erectoides phenotype. This phenotype possesses much reduced internode and petiole lengths and is practically insensitive to applied GA₁, compared with Lk plants. Application of the ethylene synthesis inhibitor, aminoethoxyvinylglycine (AVG), resulted in increased internode elongation and increased GA-sensitivity in lk plants, but not in the Lk line, L53. The ethylene-releasing compound, ethephon, had the reverse effect when applied to the Lk line, L58, reducing internode length and GA-sensitivity. Ethylene production was higher in lk segregates than in Lk segregates under the conditions used, and the shoot anatomy of lk segregates was consistent with these higher ethylene levels.
These results suggest that the phenotypic effects of gene lk may be due, at least in part, to increased ethylene production in erectoides plants. However, AVG application to lk plants did not produce true phenocopies of comparable lk plants and ethephon application to Lk plants did not produce the erectoides phenotype. Further work is therefore required to determine whether the effect of the gene lk on ethylene production is the primary action of this gene or merely a secondary consequence.     

Internode length in Pisum. Two further mutants, lh and ls, with reduced gibberellin synthesis, and a gibberellin insensitive mutant, lk

Three further internode length mutants in peas ( Pisum sativum L.), lh , ls and lk , were examined to determine if they influenced gibberellin synthesis or sensitivity. Two mutants, lh and ls , showed pronounced elongation in response to applied GA₁ and extracts from their shoots contained little gibberellin-like activity when assayed on the rice seedling (cv. Tan ginbozu) bioassay compared with similar extracts from essentially isogenic Lh and Ls plants. The third mutant, lk , was almost insensitive to applied GA₁ and at no dose rate did it become a phenocopy of normal Lk plants. Extracts from the shoots of lk and Lk segregants contained similar levels of gibberellinlike substances. All three mutants influenced growth in both the light and the dark, although only the effect of genes Lh and Ls were graft transmissible. These results suggest that lh and ls are mutants with reduced gibberellin synthesis, while lk is the first gibberellin-insensitive dwarfing gene identified in peas.     

Internode length in Pisum

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [¹³C, ³H]GA₂₀. The substrate was metabolised to [¹³C, ³H]GA₁, [¹³C, ³H]GA₈ and [¹³C, ³H]GA₂₉ in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [¹³C, ³H]GA₂₉ and, in one line, [¹³C, ³H]GA₂₉-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3-hydroxylation of GA₂₀ to GA₁. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [¹³C, ³H]GA₂₀ metabolites were considerably diluted by endogenous [¹²C]GAs, implying that the metabolism of [¹³C, ³H]GA₂₀ mirrored that of endogenous [¹²C]GA₂₀. In contrast, the [¹³C, ³H]GA₂₀ metabolites in na lines showed no dilution with [¹²C]GAs, confirming that the na mutation prevents the production of C₁₉-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the ¹²C:¹³C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [¹³C, ³H]GA₂₀ and extraction. No [¹²C]GA₁ and only traces of [¹²C]GA₈ (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Internode length in Pisum. Gibberellins and the slender phenotype

Pea plants ( Pisum sativum L.) possessing the slender phenotype (conferred by the gene combination la cry^s ) have extremely long, thin internodes and are phenotypically similar to dwarf plants (possessing genes La and/or Cry ) that have been treated with a non-limiting dose of gibberellin (GA₃). In contrast to tall and dwarf plants, slender plants are virtually insensitive to treatment with AMO 1618, PP333 or GA₃ and addition of the "gibberellin-less" mutant gene na does not alter the phenotype of slender plants. Na slender segregates possessed lower levels of gibberellin-like substances than comparable dwarf segregates when extracts from shoots were assayed using the lettuce hypocotyl or rice seedling bioassays. In addition, na slenders possessed little or no gibberellin-like activity even though they possessed a slender phenotype. Thus the gene combination la cry^s causes slender plants to respond as if they are saturated with gibberellins for growth. In addition, the gene combinations la cry^s and le la cry^c (allele cry^c is less extreme in effect than cry^s ) are shown to be almost completely epistatic to the alleles at the na locus. All these results suggest that gibberellin levels are not important in determining the internode length of slender peas (genotype la cry^s ). The possible mechanisms by which this could occur are discussed.     

Internode length in Pisum. The lrs mutation reduces gibberellin response and levels

We describe a new mutation, lrs , which reduces internode length in Pisum sativum L. The mutation appears to act by reducing both GA synthesis and the response to GA₁. The levels of the 13‐hydroxylated GAs, GA₅₃, GA₄₄, GA₁₉, GA₂₀, GA₁, and GA₈ in the lrs mutant were greatly reduced compared with the wild‐type. The extent of the reduction in GA₁ content in the apical tissues would, at least in part, account for the dwarf phenotype of the mutant. The reduced GA responsiveness of the new mutant was indicated by the inability of applied GA₁ to remove the difference in elongation between lrs and LRS plants. The lrs mutant appears to be unique amongst internode length genotypes, possessing characteristics of both GA synthesis and GA response mutants.     

Internode length in Pisum. IV. The effect of the Le gene on gibberellin metabolism

The highly active, polar gibberellin-like substance found in the apical region of shoots of tall (genotype Le ) peas ( Pisum sativum L.) is shown by combined gas chromatography-mass spectrometry (GC/MS) to be GA₁. This substance is either absent or present at only low levels in dwarf ( le ) plants. Multiple ion monitoring (MIM) tentatively suggests that GA₈ may also be present in shoot tissue of tall peas. Gibberellin A₁ is the first 3 β-hydroxylated gibberellin positively identified in peas, and its presence in shoot tissue demonstrates the organ specificity of gibberellin production since GA₁ has not been detected in developing seeds. Application of GA₁ can mask the Le/le gene difference. However, whilst Le plants respond equally to GA₂₀ and GA₁, le plants respond only weakly to GA₂₀, the major biologically active gibberellin found in dwarf peas. These results suggest that the Le gene controls the production of a 3 β-hydroxylase capable of converting GA₂₀ to GA₁. Further support for this view comes from feeds of [³H] GA₂₀ to Le and le plants. Plants with Le metabolise [³H] GA₂₀ to three major products whilst le plants produce only one major product after the same time. The metabolite common to Le and le plants co-chromatographs with GA₂₉. The additional two metabolites in Le peas co-chromatograph with GA₁ and GA₈.     

Internode length in Lathyrus odoratus. The involvement of gibberellins

Evidence was obtained by gas chromatography-mass spectrometry and gas chromatography-selected ion monitoring for the presence of gibberellin A₂₀), GA₁, GA₂₉, GA₈ and 2-epiGA₂₉ in vegetative shoots of tall sweet pea, Lathyrus odoratus L. Both tall (genotype L –) and dwarf (genotype II ) sweet peas elongated markedly in response to exogenous GA₁ attaining similar internode lengths at the highest dose levels. Likewise internode length in both genotypes was reduced by application of the GA biosynthesis inhibitor, PP333. The ratio of leaflet length to width was reduced by application of PP333 to tall plants and this effect was reversed by GA₁. When applied to plants previously treated with PP333, GA₂₀ promoted internode elongation of L – plants as effectively as GA₁, but GA₂₉ was not as effective as GA₁ when applied to II plants. In contrast, GA₂₀ and GA₁ were equally effective when applied to the semidwarf lb mutant but GA-treated lblb plants did not attain the same internode length as comparable GA-treated Lb – plants. The difference in stature between the tall and dwarf types persisted in dark-grown plants. It is concluded that GA₁ may be important for internode elongation and leaf growth in sweet pea. Mutant l may influence GA₁ synthesis by reducing 3β-hydroxylation of GA₂₀ whereas mutant lb appears to affect GA sensitivity.     

Internode length in Lathyrus odoratus. Effects of mutants l and lb on gibberellin metabolism and levels

After the application of [¹³C³H]-gibberellin A₂₀ to wild-type (tall) sweet peas ( Lathyrus odoratus L.) labelled gibberellin A₁ (GA₁), GA₈, GA₂₉ and 2-epiGA₂₉ were identified as major metabolities by gas chromatography-mass spectrometry after high performance liquid chromatography. By contrast in genetically comparable dwarf ( II ) plants only labelled GA₂₉ and 2-epiGA₂₉ were produced in significant amounts, although evidence was obtained for trace amounts of labelled GA₁ and GA₈. The apical portions of dwarf plants contained 8–10 times less GA₁ than those of tall plants but at least as much GA₂₀ (measured using di-deuterated internal standards). In conjunction with previous data these results strongly indicate that in genotype ll internode length is reduced and leaf growth altered by a reduction in GA1 levels attributable to a partial block in the 3β-hydroxylation of GA₂₀ to GA₁.
In contrast to dwarf plants, semidwarf plants (genotype lblb ) contained more GA₁ in the apical portion than wild-type counterparts. This is consistent with the suggestion that lb alters some aspect of GA sensitivity.     

Internode length in Pisum. Gene lkd

A new, single gene, recessive internode length mutant in Pisum, lkd, is described. The internodes of lkd plants are ca. 40% shorter than comparable Lkd plants and this difference appears greater in the dark than in the light. The mutant does not appear to be dwarfed due to modified gibberellin (GA) levels, as determined by gas-chromatography-selected ion monitoring (GC-SIM) for GA₁ and GA₂₀. In relative terms, the mutant responds as well as the wild-type to applied GA₁. However due to its initial short stature it does not elongate to the same extent as the wild-type to high doses of GA₁ suggesting that some other factor, unrelated to GA levels or perception is probably limiting growth in this mutant. Author for correspondence     

Internode length in Zea mays L.

[¹³C, ³H]Gibberellin A₂₀ (GA₂₀) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [¹³C, ³H]Gibberellin A₂₀ was metabolized to [¹³C, ³H]GA₂₉-catabolite and [¹³C, ³H]GA₁ by the normal, and to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₁ by the dwarf-5 mutant. In the dwarf-1 mutant, [¹³C, ³H]GA₂₀ was metabolized to [¹³C, ³H]GA₂₉ and [¹³C, ³H]GA₂₉-catabolite; no evidence was found for the metabolism of [¹³C, ³H]GA₂₀ to [¹³C, ³H]GA₁. [¹³C, ³H]Gibberellin A₈ was not found in any of the feeds. In all feeds no dilution of ¹³C in recovered [¹³C, ³H]GA₂₀ was observed. Also in the dwarf-5 mutant, the [¹³C]label in the metabolites was apparently undiluted by endogenous [¹³C]GAs. However, dilution of the [¹³C]label in metabolites from [¹³C, ³H]GA₂₀ was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA₂₀ to GA₁.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

Internode length and anatomical changes in Pisum genotypes crys and cryc in response to extended daylength and applied gibberellin A1

Dwarf pea (Pisum sativum L.) plants with genotypes cry^c and cry^s responded differently when an 8 h photoperiod (8 h daylight, 16 h dark) was extended to 24 h (8 h daylight, 16 h incandescent light). Genotype cry^c showed up to a 4-fold increase in internode length, sustained by increases in both cell length (particularly of epidermal cells) and cell number (particularly of cortical cells) while cry^s plants showed up to a 2-fold increase in internode length sustained mostly by an increase in cell number. Under an 8 h (daylight) photoperiod the two genotypes did not differ in their sensitivity to applied gibberellin A₁ (GA₁) and they showed a similar pattern of response. GA₁ significantly increased internode length, cell length and cell number in both genotypes. Incandescent light did not increase the size of the response to GA₁ except for cry^s plants at high dose rates of GA₁ (29–58 nmol). At saturating doses of GA₁ the two genotypes attained a similar peak internode length; incandescent light increased the peak by about 40%. GA₁ increased the rate of leaf appearance by up to 33% while incandescent light reduced the rate by 4–7%. The elongation response of the more mature internodes of cry^c plants to GA₁ or incandescent light was due primarily to an increase in cell length whereas increased cell number made a significant contribution in the case of internodes which were relatively immature at the time the stimulus was applied. The progressive increase in internode length of both genotypes during ontogeny was due primarily to an increase in cell number. In conclusion, alleles cry^c and cry^s (background le La) do not confer a difference in sensitivity to GA₁ and the increase in internode length in response to incandescent light is probably not the result of a real or perceived increase in GA₁ level. Allele cry^s may partially block a phytochrome mediated response to light and the key difference between genotypes cry^s and cry^c may lie in the greater elongation (extensibility?) of cry^c epidermal cells in incandescent light.     

Internode length in Pisum. III The effect and interaction of the Na/na and Le/le gene differences on endogenous gibberellin-like substances

In the garden pea ( Pisum sativum L.), shoots of the extremely short plants with the mutant na (phenotype nana) are found by bioassay to contain undetectable levels of gibberellin-like substances. This is confirmed by the use of near isogenic lines differing at the Na locus. Thus, mutant na appears to block a step early in the pathway of gibberellin synthesis. It is suggested that the polar gibberellin-like substance found in the apical portion of shoots of tall ( Le ) but not dwarf ( le ) peas could be GA₁. Extracts of shoots of na Le peas treated with GA₂₀ (the major active gibberellin in dwarf peas) possess a large amount of GA₁-like activity whereas extracts of shoots of na le peas treated with GA₂₀ possess a much reduced amount. Thus, gene Le may allow the conversion of a less active gibberellin (GA₂₀) into one more active in stimulating elongation in the pea (the GA₁-like compound). In contrast to their influence in the shoot, the na and Le genes do not appear to be operative in controlling the gibberellin content of developing seed, indicating that organ specific gibberellin biosynthesis and metabolism occur in peas.     

Gene expression during two alternative pathways of ovary development in Pisum sativum: fruit development and ovary senescence

Pea ovaries are induced to enter a fruit development pathway involving physiological and morphological changes by pollination or application of plant growth regulators. In the absence of these stimuli, overies stop growing and enter an alternative pathway of senesecence that leads to their degeneration. We have used two dimensional polyacrylamide gel electrophoresis in search of molecular changes underlying fruit development and ovary senescence at the level of total accumulated proteins, newly synthesized proteins, and translatable, RNA populations. We have found changes in gene expression during the processes of ovary formation and ovary senescence. Stimuli that induce fruit set do not appreciably alter the overall patterns of synthesized proteins or translatable RNAs, indicating that fruit development is apparently a natural continuation of ovary formation. However, ovary senescence is an alternative pathway that involves the presence of new RNA messengers and proteins as well as the disappearance of others. These changes were detected earlier than any morphological or structural changes could be observed in the ovary.