Dehiscence of Fruit in Oilseed Rape (Brassica nap us L.): II. THE ROLE OF CELL WALL DEGRADING ENZYMES AND ETHYLENE

Pod shatter in oilseed rape is accompanied by the degradationof the cell wall at the site of fruit dehiscence. Cell separationis preceded by an increase in the activity of the hydrolyticenzyme cellulase (ß 1,4-glucanase, E.C. 3.1.2.4 [EC] ),and this rise in enzyme activity is restricted to the dehiscencezone cells. In contrast, the activity of the cell wall degradingenzyme polygalacturonase (E.C. 3.2.1.15 [EC] ) exhibits no correlationeither temporally or spatially with pod dehiscence. An analysisof the ethylene production profile by intact pods during maturationhas revealed the existence of an ethylene climacteric and thisis temporally correlated with the tissue-specific increase incellulase activity. The major site of ethylene production bythe fruit has been identified to be the developing seed. Sincemaintenance of intact pods in ethylene accelerates both thesenescence and dehiscence of the tissue, it is possible thatthis gaseous regulator plays an important role in the processof pod shatter in vivo. Key words: Oilseed rape, Brassica napus, pod shatter, cellulase, polygalacturonase, ethylene     

Anatomical and Biochemical Changes Associated with the Induction of Oilseed Rape (Brassica napus) Pod Dehiscence by Dasineura brassicae (Winn.)

Dehiscence of pods at an early stage of development is a characteristicof oilseed rape pods damaged by Dasineura brassicae (pod midge).Anatomical examination of pods exhibiting symptoms of infestationrevealed a loss of cohesion between intact cells of the dehiscencezone, a narrow tissue of thin-walled cells present between thevalve margins. Determination of hydrolytic enzyme activity inpericarp tissues of damaged siliquae showed localized enhancementof both polygalacturonase (EC 3.2.1.15 [EC] ) and cellulase (ß1,4 glucanase, EC 3.1.2.4 [EC] ) activity, positionally consistent withthese factors being responsible for the observed cell wall degradation.Mechanistic similarities of midge-induced and maturation-associatedpod dehiscence are discussed. Oilseed rape, Brassica napus L. cv., Bienvenu, pod shatter, pod midge, Dasineura brassicae (Winn.), cellulase, polygalacturonase     

The role of auxin in cell separation in the dehiscence zone of oilseed rape pods

Concentrations of both free and conjugated indole-3-acetic acid(IAA) were studied during development of pod wall, dehiscencezone and seeds of Brassica napus pods. A decrease in auxin contentprior to moisture loss in the pods was observed specificallyin the dehiscence zone, which was correlated with a tissue specificincrease in ß-1,4-glucanase activity. Furthermore,treatment of the pods with the auxin mimic 2-methyl-4-chlorophenoxyaceticacid resulted in a delay of 10 d of ß-1,4-glucanaseactivity and con-comitant cell separation in the dehiscencezone. This indicates that the activity of hydrolytic enzymesinvolved in cell separation in the dehiscence zone is regulatedby auxin activity. Comparison of parthenocarpic pods with seeded pods pointed tothe seeds as the source of IAA. Levels in the dehiscence zoneof these pods were low over the entire sampling period, whilecell separation in the dehiscence zone was delayed by about4 d. These results indicate that a low level of auxins in thedehiscence zone is necessary for dehiscence to take place, butother factors may also be important. Key words: Brassica napus, pod dehiscence, auxin, cellulase     

Ethylene biosynthesis in oilseed rape pods in relation to pod shatter

Ethylene production was studied during the development and senescence of seeds and pericarp tissues of oilseed rape (Brassica napus L.) pods (siliquae). In the course of the rise to a pre-senescence climacteric, little change in 1-aminocyclopropane-1-carboxylic acid (ACC) was recorded in the seeds, indicating a rapid conversion to ethylene. In contrast, very small amounts of ethylene were produced by the pod wall (PW) tissues, which included the dehiscence zone (DZ), while levels of free and conjugated ACC in the PW increased consistently. As climacteric thylene production by the seeds declined, biosynthesis of ethylene by the PW increased. Effects of reducing ethylene production by various means were examined in relation to cell separation in the dehiscence zone. Aminoethoxyvinylglycine (AVG) applied during the pre-senescence climacteric reduced ACC levels and ethylene production by the seeds, but did not affect subsequent values in the PW. The production of -1,4-glucanase and the separation of the cells of the DZ were delayed for 3-4 d by AVG, but the force required to open fully mature pods was unaltered. In parthenocarpic (seedless) pods, ethylene was produced during senescence. Cell separation in the DZ took place as in seeded pods, although it was also delayed by 3-4 d. The results are related to changes in indole-3-acetic acid (IAA) levels in oilseed rape pods which decline in PW and DZ tissues during senescence. It is concluded that separation in the cells of the dehiscence zone requires only small amounts of ethylene to trigger the process when IAA levels are low.     

Cell Separation Processes in Plants--Models, Mechanisms and Manipulation

Abscission and dehiscence are developmental processes that involvethe co-ordinated breakdown of the cell wall matrix at discretesites and at specific stages during the life cycle of a plant.In this review we examine the events that influence the differentiationof abscission and dehiscence zone cells and the changes thatare associated with wall degradation. There is convincing evidenceto believe that ethylene and auxin co-ordinate the timing ofleaf, flower and fruit abscission but the events that regulatedehiscence and seed abscission are unclear. The use of transgenicplants and model systems such as Arabidopsis is assisting ourunderstanding of the mechanisms that regulate abscission anddehiscence and the application of this information will advanceour understanding of cell separation processes in general. Armedwith this knowledge it should be possible to either delay oraccelerate abscission and dehiscence, and this could have majorbenefits for the agricultural and horticultural industries.Copyright 2000 Annals of Botany Company Abscission, dehiscence, cell separation, wall degradation, gene expression, polygalacturonase, ß-1,4-glucanase, pathogenesis-related proteins, ethylene     

In Vivo Fixation of CO2 by Attached Pods of Brassica campestris L.

In vivo net CO₂ exchange characteristics of attached Brassicapods were studied during the entire period of their growth anddevelopment after anthesis. ¹⁴CO₂ was fed both from the externalatmosphere and internally through the pod cavity, and the anatomyof the pod-wall was examined microscopically. Stomata were observedin the outer epidermal layer of the pod wall. Net in vivo CO₂fixation by the pods was observed throughout the period of theirdevelopment and was maximum on day 42 after anthesis (DAA).Compared to the internal feeding experiments, ¹⁴CO₂ fixationfrom the external environment was very high. Apparent translocationof fixed carbon from the pod wall to seeds was rapid. Pod photosynthesiscontributed substantially to seed growth. pods, Brassica campestris L, CO₂ fixation, stomata     

In Vivo Fixation of CO2 by Attached Pods of Brassica campestris L.

In vivo net CO₂ exchange characteristics of attached Brassicapods were studied during the entire period of their growth anddevelopment after anthesis. ¹⁴CO₂ was fed both from the externalatmosphere and internally through the pod cavity, and the anatomyof the pod-wall was examined microscopically. Stomata were observedin the outer epidermal layer of the pod wall. Net in vivo CO₂fixation by the pods was observed throughout the period of theirdevelopment and was maximum on day 42 after anthesis (DAA).Compared to the internal feeding experiments, ¹⁴CO₂ fixationfrom the external environment was very high. Apparent translocationof fixed carbon from the pod wall to seeds was rapid. Pod photosynthesiscontributed substantially to seed growth. pods, Brassica campestris L., CO₂ fixation, stomata     

Genome-Wide Delineation of Natural Variation for Pod Shatter Resistance in Brassica napus

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.     

Increased resistance to pod shatter is associated with changes in the vascular structure in pods of a resynthesized Brassica napus line

The architecture of the pod wall and dehiscence zone (DZ) was studied in populations of a resynthesized, shatter-resistant, oilseed rape line, DK142, and the commercial cultivar Apex. The dimensions of the pod wall and its component tissues were significantly larger in DK142. However, the variation in the pod architecture of Apex, DK142 and F2 populations derived from crosses of DK142 and Apex was found to have little or no role in pod shatter. By contrast, variation in the dimensions of the DZ characters correlated strongly and positively with shatter resistance. The size of the main vascular bundle (MVBV) of DK142 as it exited the valve and joined the vascular tissue of the replum was, on average, 60% larger than in Apex, the DZ was 40% wider and there was a high preponderance of vascular tissue other than the MVBV. The variation in the size of the MVBV accounted for much of the variation in shatter resistance of all populations, including shatter-susceptible Apex. The DZ width was also found to be important in explaining the limited range of shatter values in Apex, but in populations of DK142 and F2, where the amount of vascular intrusion into the DZ was much greater, the variation in DZ width was not important. The importance of the vascular tissue to shatter resistance was further highlighted by a novel microfracture test (MFT). By contrast, no significant difference between DK142 and Apex in the ease of separation of the thin-walled DZ cells was detected using the MFT.     

Low Temperature Affects Pattern of Leaf Growth and Structure of Cell Walls in Winter Oilseed Rape (Brassica napus L., var. oleifera L.)

Three-week acclimation of winter oilseed rape (Brassica napusL. var. oleifera L.) plants in the cold (2 °C) resultedin a modified pattern of leaf cell enlargement, indicated bythe increased thickness of young leaf blades and modified dimensionsof mesophyll cells, as compared with non-acclimated tissuesgrown at 20/15 °C (day/night). The thickness of leaf cellwalls also increased markedly during cold acclimation but itdecreased in response to a transient freezing event (5 °Cfor 18 h followed by 6 or 24 h at 2 °C, in the dark). Cellwalls of the upper (adaxial) epidermis were most affected. Theirultrastructure was modified by cold and freezing treatmentsin different ways, as revealed by electron microscopy. Possiblereasons for the cold- and freezing-induced modifications inthe leaf and cell wall morphology and their role in plant acclimationto low temperature conditions are discussed. Copyright 1999Annals of Botany Company Acclimation, Brassica napus var. oleifera, cell wall ultrastructure, cold, freezing, leaf structure, winter oilseed rape.     

Ovule Fertilization and Seed Number per Pod Determination in Oil Seed Rape (Brassica napus)

Ovule fertilization in relation to seed number determinationin spring rapeseed, Brassica napus var. Maris Haplana, was investigated.Failure of fertilization was found to be the major factor limitingthe number of seeds per pod in both the basally and apicallylocated pods on the terminal inflorescence. Sufficient pollengermination on a stigma did not guarantee full seed set andeven when pollen tubes were present at the micropyle regions,ovules were not penetrated. Some seeds aborted: in some, triplefusion did not take place while other seeds were small and deemedat disadvantage to compete for available nutrients. Brassica napus, fertilization, ovules, seeds     

Defoliation and its Effects on Pod and Seed Development in Oil Seed Rape (Brassica napus L.)

Both in vivo and in vitro techniques have been used to followthe development of individual pods on the terminal inflorescenceof undefoliated and defoliated plants of oil-seed rape (Brassicanapus cv. Maris Haplona). For any pod, a rapid increase in podlength occurred between 2 d and 8 d after flower opening andthis preceded by approximately 2 d the increase in pod width,the rate of which was less than that for length. An increasein the diameter of individual seeds coincided with the increasein pod width. Regional increases in the length and width ofpods were associated with the presence of developing seeds inthese regions. Key words: Brassica napus L., Development, In vivo, Pod and seed, Stress     

Histology of Shoot Bud Ontogeny from Seedling Root Segments of Brassica napus L.

Root explants of Brassica napus cultured in vitro form adventitiousshoots. The root buds originated at the base of the newly initiatedlateral root. Cells in association with the differentiatingphloem of the developing lateral roots were the sites for rootbud formation. A nodular mass of cytoplasmic cells developedby day 7 at the base of the lateral root. This group of cellscontinued to divide an enlarge. The cells in the peripheralregion of the nodular cell mass differentiated further intoa meristematic zone. The meristematic cells grew towards theperiphery of the cortex by crushing the outer layer of corticalcells. Further development of the meristematic layer resultedin the formation of shoot primordia with organized shoot apicalmeristems and leaf primordia.Copyright 1993, 1999 Academic Press Brassica napus, canola, cultured root segments, root buds     

Localization of Phenolic Compounds in the Root Cap Columella of Six-year-old Dry Seeds of Brassica napus during Imbibition and Germination

In 6-year-old seeds of Brassica napus the columella cells haveno necroses and resemble in structure the cells of the 2-year-oldembryo. The outermost layer of the columella shows a structuresimilar to that of the lateral region of the root cap, as itcontains protein bodies, rare in layers of the columella closerto the promeristem, which, in turn, contain numerous mitochondriaand plastids. Phenolic compounds in the dry embryo are on thesurface of the root cap in the space between the plasmalemmaand the cell wall, and in small vesicles which presumably remainedfrom degradation of ER. Imbibition promotes further extrusionof phenolics outside the plasma membrane. Long sheets of ERare visible after 9 h imbibition. After 24 h phenolics of moredense structure are localized in some dilated parts of the ER.This suggests that new production of defence compounds startswithin 24 h in water, a few hours earlier than in 2-year-oldseeds.Copyright 1994, 1999 Academic Press Brassica napus, phenolics, root columella, germination     

The effect of <Emphasis Type="Italic">INDEHISCENT</Emphasis> point mutations on silique shatter resistance in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis>)

Key message

This study elucidates the influence of indehiscent mutations on rapeseed silique shatter resistance. A phenotype with enlarged replum-valve joint area and altered cell dimensions in the dehiscence zone is described.

Abstract

Silique shattering is a major factor reducing the yield stability of oilseed rape (Brassica napus). Attempts to improve shatter resistance often include the use of mutations in target genes identified from Arabidopsis (Arabidopsis thaliana). A variety of phenotyping methods assessing the level of shatter resistance were previously described. However, a comparative and comprehensive evaluation of the methods has not yet been undertaken. We verified the increase of shatter resistance in indehiscent double knock-down mutants obtained by TILLING with a systematic approach comparing three independent phenotyping methods. A positive correlation of silique length and shatter resistance was observed and accounted for in the analyses. Microscopic studies ruled out the influence of different lignification patterns. Instead, we propose a model to explain increased shattering resistance of indehiscent rapeseed mutants by altered cell shapes and sizes within the contact surfaces of replum and valves.