Delay of early fruitlet abscission by branch girdling in citrus coincides with previous increases in carbohydrate and gibberellin concentrations

Current evidence in citrus indicates that gibberellins (GAs) are main determinants of early fruit set while subsequent growth of developing fruits is mostly dependent upon carbohydrate availability. In this work, branch girdling performed at anthesis in Satsuma mandarin (Citrus unshiu (Mak.) Marc.) cv. Okitsu transitorily reduced early abscission rates (12–32 days after anthesis, DAA) delaying initially the process of natural fruitlet drop. The effects of girdling on growth, gibberellin (GA) and carbohydrate concentrations in developing ovaries and fruitlets were assessed during this initial growth stage (0–69 DAA). In girdled branches, abscission rate reduction was preceded by elevated concentrations of carbohydrate and GA in developing ovaries and fruitlets. Girdling at anthesis stimulated higher hexose (21 DAA) and starch (6–20 DAA) concentrations and also higher GA₁ (6 DAA), GA₁₉ (13–20 DAA) and GA₂₀ (6–20 DAA). The results established a relationship between the reduction of early abscission rates and higher concentrations of carbohydrates and GAs induced by girdling in developing fruitlets. These findings revealed that girdling certainly increased GA concentration and strongly suggested that its effect on early fruitlet abscission delay is likely mediated by both GA and carbohydrates.     

Activation of gibberellin 20-oxidase 2 undermines auxin-dependent root and root hair growth in NaCl-stressed <Emphasis Type="Italic">Arabidopsis</Emphasis> seedlings

Although salt stress mainly disturbs plant root growth by affecting the biosynthesis and signaling of phytohormones, such as gibberellin (GA) and auxin, the exact mechanisms of the crosstalk between these two hormones remain to be clarified. Indole-3-acetic acid (IAA) is a biologically active auxin molecule. In this study, we investigated the role of Arabidopsis GA20-oxidase 2 (GA20ox2), a final rate-limiting enzyme of active GA biosynthesis, in IAA-directed root growth under NaCl stress. Under the NaCl treatment, seedlings of a loss-of-function ga20ox2-1 mutant exhibited primary root and root hair elongation, altered GA₄ accumulation, and decreased root Na⁺ contents compared with the wild-type, transgenic GA20ox2-complementing, and GA20ox2-overexpression plant lines. Concurrently, ga20ox2-1 alleviated the tissue-specific inhibition of NaCl on IAA generation by YUCCAs, IAA transport by PIN1 and PIN2, and IAA accumulation in roots, thereby explaining how NaCl increased GA20ox2 expression in shoots but disrupted primary root and root hair growth in wild-type seedlings. In addition, a loss-of-function pin2 mutant impeded GA20ox2 expression, indicating that GA20ox2 function requires PIN2 activity. Thus, the activation of GA20ox2 retards IAA-directed primary root and root hair growth in response to NaCl stress.     

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA₂₀ to bioactive GA₁) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₂₀ to GA₂₉), suggesting a concerted regulation to increase levels of bioactive GA₁ following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₁ to GA₈) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA₁, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [¹⁴C]GA₁₂ to [¹⁴C]GA₁ only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA₁ required for initial fruit set and growth.     

Photoperiodic flower induction in <Emphasis Type="Italic">Ipomoea nil</Emphasis> is accompanied by decreasing content of gibberellins

The involvement of gibberellins (GAs) in the control of flower induction in the short-day plant Ipomoea nil has been investigated. To clarify the molecular basis of this process, we identified the full-length cDNAs of the InGA20ox3 and InGA2ox1 genes, which encode enzymes responsible for GA biosynthesis and catabolism, respectively. We studied the expression patterns of both genes and determined the tissue and cellular immunolocalisation of gibberellic acid (GA₃) in the cotyledons of 5-day-old seedlings growing under inductive and non-inductive photoperiodic conditions. In the second half of the inductive night, which is crucial for flower induction in I. nil, InGA20ox3 expression decreased, whereas InGA2ox1 mRNA accumulated, which indicates that photoperiod regulates the activity of both genes. Furthermore, these changes are correlated with GA₃ level. Thus, our results support the thesis that the proper balance between the expression of the InGA20ox3 and InGA2ox1 genes and low GA₃ content correlate with photoperiodic flower induction in I. nil.     

Engineering gibberellin metabolism in <Emphasis Type="Italic">Solanum nigrum</Emphasis> L. by ectopic expression of gibberellin oxidase genes

Gibberellins (GAs) control many aspects of plant development, including seed germination, shoot growth, flower induction and growth and fruit expansion. Leaf explants of Solanum nigrum (Black Nightshade; Solanaceae) were used for Agrobacterium-mediated delivery of GA-biosynthetic genes to determine the influence of their encoded enzymes on the production of bioactive GAs and plant stature in this species. Constructs were prepared containing the neomycin phosphotransferase (nptII) gene for kanamycin resistance as a selectable marker, and the GA-biosynthetic genes, their expression under the control of the CaMV 35S promoter. The GA-biosynthetic genes comprised AtGA20ox1, isolated from Arabidopsis thaliana, the product from which catalyses the formation of C₁₉-GAs, and MmGA3ox1 and MmGA3ox2, isolated from Marah macrocarpus, which encode functionally different GA 3-oxidases that convert C₁₉-GAs to biologically active forms. Increase in stature was observed in plants transformed with AtGA20ox1, MmGA3ox2 and MmGA3ox1 + MmGA3ox2, their presence and expression being confirmed by PCR and RT-PCR, respectively, accompanied by an increase in GA₁ content. Interestingly, MmGA3ox1 alone did not induce a sustained increase in plant height, probably because of only a marginal increase in bioactive GA₁ content in the transformed plants. The results are discussed in the context of regulating plant stature, since this strategy would decrease the use of chemicals to promote plant growth.     

Characterization of <Emphasis Type="Italic">GA20ox</Emphasis> genes in tall and dwarf types coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconuts (Cocos nucifera L.) are divided by the height into tall and dwarf types. In many plants the short phenotype was emerged by mutation of the GA20ox gene encoding the enzyme involved in gibberellin (GA) biosynthesis. Two CnGA20ox genes, CnGA20ox1 and CnGA20ox2, were cloned from tall and dwarf types coconut. The sequences, gene structures and expressions were compared. The structure of each gene comprised three exons and two introns. The CnGA20ox1 and CnGA20ox2 genes consisted of the coding region of 1110 and 1131 bp, encoding proteins of 369 and 376 amino acids, respectively. Their amino acid sequences are highly homologous to GA20ox1 and GA20ox2 genes of Elaeis guineensis, but only 57% homologous to each other. However, the characteristic amino acids two histidines and one aspartic acid which are the two iron (Fe²⁺) binding residues, and arginine and serine which are the substrate binding residues of the dioxygenase enzyme in the 20G-FeII_Oxy domain involved in GA biosynthesis, were found in the active site of both enzymes. The evolutionary relationship of their proteins revealed three clusters in vascular plants, with two subgroups in dicots and three subgroups in monocots. This result confirmed that CnGA20ox was present as multi-copy genes, and at least two groups CnGA20ox1 and CnGA20ox2 were found in coconut. The nucleotide sequences of CnGA20ox1 gene in both coconut types were identical but its expression was about three folds higher in the leaves of tall coconut than in those of dwarf type which was in good agreement with their height. In contrast, the nucleotide sequences of CnGA20ox2 gene in the two coconut types were different, but the expression of CnGA20ox2 gene could not be detected in either coconut type. The promoter region of CnGA20ox1 gene was cloned, and the core promoter sequences and various cis-elements were found. The CnGA20ox1 gene should be responsible for the height in coconut, which is different from other plants because no mutation was present in CnGA20ox1 gene of dwarf type coconut.     

Carbohydrate Availability in Relation to Fruitlet Abscission in Citrus

Abscission of flowers and fruitlets in the Washington navelorange (Citrus sinensis [L.] Osbeck) has been characterizedin relation to carbohydrate availability. A main wave of flowerabscission occurs shortly after anthesis while the carbohydratereserves in the tree are high. Fruitlet abscission starts approx.30 d after the commencement of flowering, while carbohydrates(mainly starch) are being accumulated in the leaves. Flowerand early fruitlet abscission are not caused by carbohydrateshortage. During late fruitlet abscission sucrose concentrationin the leaves falls to a low value demonstrating a limitationin supply and competition among the developing fruitlets forcarbohydrates. Concentrations of sucrose and reducing sugarsin the peel of the fruitlets also fall to low values, and arelationship could be demonstrated between these free sugarlevels and abscission. Ringing increases carbohydrate supplyto fruit and reduces late fruitlet abscission, but only hasa marginal effect on the growth of the fruitlets, which seemsless sensitive than abscission to carbohydrate shortage. Thelimitation of carbohydrate supply to the fruitlets occurs whilestarch levels in the leaves remain high. Slow mobilization ofstarch reserves may be one factor limiting set in Citrus. Copyright2001 Annals of Botany Company Carbohydrate supply, citrus, fruit growth and abscission, ringing, navel orange, starch, sugar metabolism     

Auxin-Gibberellin Interactions in Pea: Integrating the Old with the New

Recent findings on auxin-gibberellin interactions in pea are reviewed, and related to those from studies conducted in the 1950s and 1960s. It is now clear that in elongating internodes, auxin maintains the level of the bioactive gibberellin, GA₁, by promoting GA₁ biosynthesis and by inhibiting GA₁ deactivation. These effects are mediated by changes in expression of key GA biosynthesis and deactivation genes. In particular, auxin promotes the step GA₂₀ to GA₁, catalyzed by a GA 3-oxidase encoded by Mendel’s LE gene. We have used the traditional system of excised stem segments, in which auxin strongly promotes elongation, to investigate the importance for growth of auxin-induced GA₁. After excision, the level of GA₁ in wild-type (LE) stem segments rapidly drops, but the auxin indole-3-acetic acid (IAA) prevents this decrease. The growth response to IAA was greater in internode segments from LE plants than in segments from the le-1 mutant, in which the step GA₂₀ to GA₁ is impaired. These results indicate that, at least in excised segments, auxin partly promotes elongation by increasing the content of GA₁. We also confirm that excised (light-grown) segments require exogenous auxin in order to respond to GA. On the other hand, decapitated internodes typically respond strongly to GA₁ application, despite being auxin-deficient. Finally, unlike the maintenance of GA₁ content by auxin, other known relationships among the growth-promoting hormones auxin, brassinosteroids, and GA do not appear to involve large changes in hormone level.     

Hormonal regulation of fruitlet abscission induced by carbohydrate shortage in citrus

 The hormonal signals controlling fruitlet abscission induced by sugar shortage in citrus were identified in Satsuma mandarin, Citrus unshiu (Mak.) Marc, cv. Clausellina and cv. Okitsu. Sugar supply, hormonal responses and fruitlet abscission were manipulated through full, partial or selective leaf removals at anthesis and thereafter. In developing fruitlets, defoliations reduced soluble sugars (up to 98%), but did not induce nitrogen and water deficiencies. Defoliation-induced abscission was preceded by rises (up to 20-fold) in the levels of abscisic acid (ABA) and 1-aminocyclopropane-1-carboxylic acid (ACC) in fruitlets. Applications to defoliated plants showed that ABA increased ACC levels (2-fold) and accelerated fruitlet abscission, whereas norflurazon and 2-aminoethoxyvinyl glycine reduced ACC (up to 65%) and fruitlet abscission (up to 40%). Only the full defoliation treatment reduced endogenous gibberellin A₁ (4-fold), whereas exogenous gibberellins had no effect on abscission. The data indicate that fruitlet abscission induced by carbon shortage in citrus is regulated by ABA and ACC originating in the fruits, while gibberellins are apparently implicated in the maintenance of growth. In this system, ABA may act as a sensor of the intensity of the nutrient shortage that modulates the levels of ACC and ethylene, the activator of abscission. This proposal identifies ABA and ACC as components of the self-regulatory mechanism that adjusts fruit load to carbon supply, and offers a physiological basis for the photoassimilate competition-induced abscission occurring under natural conditions. Received: 19 February 1999 / Accepted: 14 August 1999     

Seed effects on gibberellin metabolism in pea pericarp

Pea fruit (Pisum sativum L.) is a model system for studying the effect of seeds on fruit growth in order to understand coordination of organ development. The metabolism of ¹⁴C-labeled gibberellin A₁₂ (GA₁₂) by pea pericarp was followed using a method that allows access to the seeds while maintaining pericarp growth in situ. Identification and quantitation of GAs in pea pericarp was accomplished by combined gas chromatography-mass spectrometry following extensive purification of the putative GAs. Here we report for the first time that the metabolism of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₉ and [¹⁴C]GA₂₀ occurs in pericarp of seeded pea fruit. Removal of seeds from the pericarp inhibited the conversion of radiolabeled GA₁₉ to GA₂₀ and caused the accumulation of radiolabeled and endogenous GA₁₉. Deseeded pericarp contained no detectable GA₂₀, GA₁, or GA₈, whereas pericarp with seeds contained endogenous and radiolabeled GA₂₀ and endogenous GA₁. These data strongly suggest that seeds are required for normal GA biosynthesis in the pericarp, specifically the conversion of GA₁₉ to GA₂₀.     

The role of gibberellins and auxin on the tomato cell layers in pericarp via the expression of ARFs regulated by miRNAs in fruit set

Phytohormones, such as auxin (IAA) and gibberellin (GA), are known to be essential for fruit development. We utilized GA-deficient (gib-3) and diageotropica (dgt) tomato mutants to elucidate the effects of single hormones in the pericarp. The application of IAA or GA, respectively, to gib-3 or dgt single mutants induced a significant morphological difference in the fruit set. We found that IAA application induced cell division in the gib-3 pericarp and that GA application did not increase the cell layers in the dgt pericarp. In molecular studies, the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were downregulated by IAA application, whereas the expression of their regulators miRNA160 and miRNA167 was upregulated by IAA application in gib-3 plants. Furthermore, the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were upregulated by GA application, whereas the expression levels of miRNAs were reduced in the dgt mutant. These results imply that the expression levels of SlARF6, SlARF8, SlARF10 and SlARF16 were negatively correlated with the number of cell layers in the pericarp during fruit set. To further support this hypothesis, 35s:mSlARF10 transgenic plants resistant to SlmiR160 cleavage of SlARF10 mRNA were used to investigate the cell layers in fruit. These results revealed that mSlARF10 overexpression indeed resulted in fewer cell layers than in wild type fruit. Together, our data suggest that GA- and IAA-mediated miRNAs and their target ARFs influence the formation of pericarp cell layers during fruit set development.     

Function and Expression Analysis of Gibberellin Oxidases in Apple

Three cDNAs, encoding gibberellin (GA) 20-oxidase (MdGA20ox1, identical to AB037114), 3-oxidase (MdGA3ox1), and 2-oxidase (MdGA2ox1), were isolated from apple cv. Fuji (Malus x domestica). Southern blot analysis indicated that each of these genes belongs to a gene family. Standard enzyme assays show that the MdGA20ox1-MBP fusion protein can sequentially oxidize three times at C-20 position of GA₁₂ and GA₅₃ and generate GA₉ and GA₂₀; the MdGA3ox1-MBP fusion protein converts GA₂₀ and GA₉ to GA₄ and GA₁, and the MdGA2ox1-MBP fusion protein converts GA₄ and GA₁ to GA₃₄ and GA₈, respectively. In addition, we confirmed that MdGA20ox1 is strongly expressed in immature seeds and scarcely detected in other tissues, whereas MdGA3ox1 and MdGA2ox1 are mainly expressed in flowers. Therefore, all the three cDNAs are localized in reproductive tissues. Functional and expression analysis of the three GA oxidases would provide fundamental molecular information to analyze GA metabolic regulation in apple.     

Changes in the accumulation of flavonoid and isoflavonoid conjugates associated with plant age and nodulation in alfalfa (Medicago sativa)

Carbohydrate and mineral nutrition was studied in relation to abscission in fruitlets from leafy inflorescences of the Washington navel orange ( Citrus sinensis [L.] Osbeck). Differences in the growth rate of the fruitlets permitted to predict abscission several weeks in advance. This allowed characterization of early differences in composition and behaviour of persisting and abscising fruitlets.
Inflorescences with persisting fruitlets accumulated more mineral elements than inflorescences with abscising fruitlets, and for the phloem-mobile elements the excess accumulation was allocated to the fruitlets. Starch accumulated in the inflorescence leaves during early fruitlet growth, and this accumulation was enhanced by the persisting fruitlets despite their higher growth rate and mobilizing ability. The relations between the fruitlets and the inflorescence leaves cannot be explained totally in terms of source sink relationships; a hormonal regulation of the leaves by the fruitlets is postulated.
Acid invertase activities and hexose concentration in the pericarp were higher in the abscising fruitlets. The lower early growth rate of these fruitlets is thus not caused by a limitation in carbohydrate supply. It seems more related to carbohydrate utilization, probably hormonally mediated, as demonstrated by the higher dependence on hormone supply for the growth in vitro of the endocarp explants.     

Identification of the dwarf gene <Emphasis Type="Italic">GmDW1</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) by combining mapping-by-sequencing and linkage analysis

Key message

GmDW1 encodes an ent-kaurene synthase (KS) acting at the early step of the biosynthesis pathway for gibberellins (GAs) and regulates the development of plant height in soybean.

Abstract

Plant height is an important component of plant architecture, and significantly affects crop breeding practices and yield. Here, we report the characterization of an EMS-induced dwarf mutant (dw) of the soybean cultivar Zhongpin 661 (ZDD23893). The dw mutant displayed reduced plant height and shortened internodes, both of which were mainly attributed to the longitudinally decreased cell length. The bioactive GA₁ (gibberellin A₁) and GA₄ (gibberellin A₄) were not detectable in the stem of dw, and the dwarf phenotype could be rescued by treatment with exogenous GA₃. Genetic analysis showed that the dwarf trait of dw was controlled by a recessive nuclear gene. By combining linkage analysis and mapping-by-sequencing, we mapped the GmDW1 gene to an approximately 460-kb region on chromosome (Chr.) 8, containing 36 annotated genes in the reference Willliams 82 genome. Of these genes, we identified two nonsynonymous single nucleotide polymorphisms (SNPs) that are present in the encoding regions of Gmdw1 and Glyma.08G165100 in dw, respectively. However, only the SNP mutation (T>A) at nucleotide 1224 in Gmdw1 cosegregated with the dwarf phenotype. GmDW1 encodes an ent-kaurene synthase, and was expressed in various tissues including root, stem, and leaf. Further phenotypic analysis of the allelic variations in soybean accessions strongly indicated that GmDW1 is responsible for the dwarf phenotype in dw. Our results provide important information for improving our understanding of the genetics of soybean plant height and crop breeding.