Citrus Fruit Development Research Articles

Partial characterization of H-translocating inorganic pyrophosphatase from 3 citrus varieties differing in vacuolar pH.

Vacuolar pyrophosphatase (V-PPase) from juice cells of 3 citrus varieties (differing in their vacuolar pH) were partially characterized using purified tonoplast vesicles. Total V-PPase activity was highest in vesicle samples from sweet limes with vacuolar pH of 5.0, while samples from acid limes (with lowest vacuolar pH of 2.0) had the minimal total V-PPase activity. Samples from 'Valencia' orange had intermediate V-PPase levels. When assayed at equal V-PPase activity (measured as Pi production), V-PPase was not able to generate a pH gradient (DeltapH) in vesicles from acid lime, despite its capacity to form a DeltapH in the presence of ATP. Vesicles from sweet lime and 'Valencia' orange were able to form similar DeltapHs in the presence of PPi and ATP supplied together or separately. Antibodies raised against a peptide corresponding to the catalytic site of mung bean V-PPase reacted with samples from all varieties, coinciding with their capacity to hydrolyze PPi. However, antibodies raised against the entire V-PPase polypeptide from mung bean recognized V-PPase from sweet lime and 'Valencia' orange, but did not recognize acid lime samples even at elevated protein concentrations. The structural differences highlighted by antibody recognition, substrate affinity and proton-pumping reactions of V-PPase presented here may reflect evolutionary adaptations related to its reduced function under in vivo conditions and are in agreement with our understanding of acid, sugar accumulation and vacuolar pH changes during the development and maturation of citrus fruits.

Molecular characterization of cDNA clones for ADP-glucose pyrophosphorylase from Citrus

Two cDNA clones encoding ADP-glucose pyrophosphorylases have been isolated from fruit and leaf cDNA libraries of Citrus ( Citrus unshiu Mac. cv. Miyagawa) in which one was designated as agpS for the small subunit and the other as agpL for the large subunit. Both cDNAs have uninterrupted open reading frames deriving 57–58 kDa polypeptides. The deduced amino acid sequence of agpS has a unique feature. That is, it lacks a cysteine residue (Cys-12) which is usually conserved in all other dicot enzymes. This is the first report of agpS lacking Cys-12 among dicot small subunits. The expression pattern of both subunits showed a different profile in which leaves synthesized both agpS and agpL more vigorously than those of fruits. During leaf development, the transcripts of agpS and agpL showed a higher expression level at younger stages. During fruit development, the expression level of both subunits was observed to be highest in the mini-green stage, but it decreased in the small green stage and it increased again towards the maturing stage. These results suggest that both subunits may play an important role in the regulation of Citrus fruit and leaf development.

Expression of a phytoene synthase gene and characteristic carotenoid accumulation during citrus fruit development

A cDNA clone encoding a protein homologue to phytoene synthase (PSY, CitPSY1) was isolated from Satsuma mandarin (Citrus unshiu Marc.) fruit. Expression analysis revealed the presence of the CitPSY1 transcript in flowers and leaves, with a higher level detected in the mature than in the young leaves. In fruit, the transcript was present in a low quantity in the flavedo (peel) and juice sacs/segment epidermis (edible part) at an early developmental stage. Later, it began to accumulate towards fruit maturation in both parts. These fluctuations were in accordance with carotenoid accumulation and chlorophyll disappearance. We suggest that CitPSY1 plays an important role in carotenoid accumulation. We also propose a possible pathway in carotenoid biosynthesis that is based on the fluctuation of the characteristic carotenoid composition during Satsuma mandarin development.

NADP +-isocitrate dehydrogenase gene expression and isozyme activity during citrus fruit development

The accumulation of citric acid and its decline toward fruit maturation is typical of citrus fruit. We studied NADP +-isocitrate dehydrogenase (NADP-IDH), an enzyme involved in citrate metabolism. A cDNA encoding the enzyme was cloned from lemon ( Citrus limon) juice sac cells, and is the first-reported NADP-IDH from fruits. Sequence comparisons and phylogenetic analysis indicate that it most probably belongs to a monophyletic clade of plant cytosolic enzymes. The mRNA level in the juice sac cells was induced during lemon fruit growth, and increased by about 15-fold to a peak as the fruit neared maturation. Spectrophotometric assay of the NADP-IDH activity in the pulp during fruit development showed that in young fruit, most of the activity was associated with the mitochondrial preparation and that, as the fruit grew, the activity shifted to the soluble fraction. The two activities could also be distinguished by isozyme gel electrophoresis: while one isozyme was detected in the mitochondrial preparation of young fruit and declined later, the other was induced in the soluble fraction of older fruit and increased as the fruit grew. The increasing activity of NADP-IDH in the soluble fraction throughout fruit development correlated well with the increase in gene expression, which suggests that the soluble activity is regulated by the expression of the cytosolic NADP-IDH gene. The possible role of this form of the enzyme in citric acid catabolism in the pulp is discussed.

Changes in the levels of mRNAs for putative cell growth-related genes in the albedo and flavedo during citrus fruit development.

Changes in mRNA levels for the seven gene homologues to endoxyloglucan transferase-related protein, expansin, extensin, β-1,3 glucanase, glycine-rich protein, pectinacetylesterase and pectinesterase, which were obtained by random sequencing studies, were investigated in relation to rind development in citrus (Citrus unshiu Marc.) fruit. Expression patterns in the albedo and flavedo were classified into four types: Type-I, transcript levels low in early fruit development but increased at the ripening stage; Type-II, transcript levels high until mid-development and then decreased towards ripening; Type-III, detectable transcript limited to fruitlets at 26 days after flowering (DAF); Type-IV, ubiquitous during the development. Based on the expression patterns, we discuss the possible roles of these genes in rind development.

Refinement of cDNA Clone Expression Analysis in Random Sequencing from the Rapid Cell Development Phase of Citrus Fruit

Summary To characterize citrus fruit development, we analyzed changes in the levels of mRNAs in nine clones that included two glycine-rich protein (GRP) genes, an expressed sequence tag (EST) clone (EST1), and six putative stress-responsive genes (pcMFR1616.41, pcMFR1804.77, pcMFR1727.28, pcMFRI729.82, pcMFRI729.148 and pcMFR1804.36) during citrus development. GRPs were of two types; one clone (CitGRPI) showed a high degree of identity to typical GRP genes, the other (CitRNA-GRP3) resembled the genes of glycine-rich RNA-binding proteins. The CitGRP 1 transcript level was high in fruitlets, flowers, and mature leaves, but decreased to low levels in the fruit during later development stages. The CitRNA-GRP3 transcript was present in all the plant organs, except mature leaves. The EST 1 transcript level was maximal in the early development stage. Expression of the six putative stress-responsive genes also was regulated distinctly, both temporally and spatially. Collectively, the findings suggest that these genes have important roles in the normal development and enlargement of citrus fruit and that they are regulated by a wide range of internal and external stimuli.

Carbohydrate Supply as a Critical Factor for Citrus Fruit Development and Productivity

Productivity is the final outcome of a long chain of developmental events. The reproductive processes leading to cropping in a fruit tree may be analyzed at two distinct levels: 1) the reproductive unit, single organ level, and 2) the multi-organ fruit population, tree level. (The orchard, with its tree population, represents a still higher organizational level.) Although a complete “failure” at the organ level precludes crop formation altogether, both types of analysis are biologically meaningful and differ in many respects. Analysis at the organ level will focus on cellular and biochemical processes. Analysis at the tree level will emphasize quantitative aspects, employing statistical and modeling approaches. Taking “fruit set” as an example, for the individual fruit it is an “all-or-none” event, governed by hormonal and biochemical systems. However, when the fruit population of an entire tree is considered, the rate of fruit set assumes a quantitative significance. The carbohydrate economy of plants, more frequently referred to as source-sink relationships, has received considerable attention in recent years, and important biochemical and regulatory aspects have been elucidated. However, such research has been conducted mainly with model plants and with a few annual crop plants. Our knowledge and understanding of these processes in fruit trees, in general, and citrus, in particular, are still very fragmentary. Carbohydrate economy may be more problematic in this respect than other research areas, because of considerable variability among plant genera and the existence of several biochemical alternatives. The source-sink physiology of citrus as related to fruit growth and productivity has been investigated extensively in the last decades, mainly at the quantitative tree level. Certain cellular and biochemical carbohydrate systems have been examined at the organ level, mainly in relation to fruit development and maturation. Most of this research has been reviewed by Goldschmidt and Koch (1996). The present article concentrates on specific aspects of citrus’ source-sink relationships that deserve further discussion, and indicates directions for future research. THE EVERGREEN FRAMEWORK

Changes in Rind Firmness and Cell Wall Polysaccharides during Citrus Fruit Development and Maturation

Changes in rind firmness and cell wall polysaccharide composition were measured in fruit with a) a soft rind, (`Satsuma' mandarin, Citrus unshiu Marc., cv. Aoshima), and b) a firm rind (hassaku, C. hassaku Hort. ex Tanaka), from August to January of the following year. Rind firmness was similar in both species in August, but hassaku had significantly firmer rind than did mandarin from September to January. Both flavedo and albedo tissues were extracted, and the extracts were hydrolyzed and fractionated to yield four fractions: (hot water, EDTA, hemicellulose, and cellulose). In flavedo tissue, sugar concentration was highest in the cellulose fraction, and lowest in the hemicellulose fraction. The concentration in all fractions decreased as the fruit developed and matured. Although the sugar concentration in the cellulose and EDTA fractions of both species was similar in August, it was significantly higher in both fractions in hassaku than in mandarin in January. The sugar concentration of each fraction from albedo tissue was in the order: cellulose > hemicellulose > hot water > EDTA. The range of variation in cell wall sugars in albedo tissue was smaller than that in flavedo tissue. Chemical name used: ethylenediaminetetraacetic acid (EDTA).

Differential expression of three sucrose-phosphate synthase isoforms during sucrose accumulation in citrus fruits ( Citrus unshiu Marc.)

The role of sucrose-phosphate synthase (SPS) in sucrose metabolism during citrus fruit development was investigated through sucrose content and enzyme activity in relation to the transcription of three SPS cDNA isoforms. The profiles of sucrose accumulation in edible tissue (juice sacs and segment epidermis) of satsuma mandarin ( Citrus unshiu Marc.) agreed with those of SPS activity, that is, an increase in sucrose content accompanied high SPS activity. However, sucrose accumulation in edible tissue preceded the increase in SPS activity. The sucrose accumulation and the activity of SPS in peel tissue (albedo and flavedo) were lower than those in edible tissue. The expressions of three SPS genes (previously isolated and designated as CitSPS1, CitSPS2, and CitSPS3) were analyzed during the fruit developmental stage. Transcripts of the CitSPS1 in edible tissue were expressed at all stages analyzed and the transcript level was highest at the mature stage (223 days after flowering (DAF)). No transcripts of the CitSPS2 were found in edible tissue before 187 DAF, but thereafter transcripts were detected and increased until the mature stage. However, transcripts of CitSPS3 were undetected in edible tissue throughout the developmental stage. The expression of CitSPS1 in peel tissue was low in young fruit, then increased notably at 148 DAF, retaining its level until the mature stage. Transcripts of CitSPS2 in peel tissue showed the same pattern as that of edible tissue. The expression of CitSPS3 in peel tissue was detected at 148, 187 and 223 DAF, although its level was weak compared with CitSPS1 and CitSPS2. The activity of SPS in edible tissue paralleled the levels of transcription of CitSPS1 and CitSPS2, suggesting that the expression of these two genes might have an important role in determining the sucrose composition or accumulation in citrus fruit. In peel tissue, the patterns of SPS activity mostly agreed with the transcripts of CitSPS1, CitSPS2 and CitSPS3. These results also indicate that SPS genes are independently regulated, resulting in distinct temporal and spatial expression patterns.

GROWTH RESPONSES FROM WHOLE FRUIT AND FRUIT HALVES OF LEMON CULTURED IN VITRO

A comparative growth study was conducted on juice vesicles cultured in the form of various fruit explant types (equatorially bissected fruit halves, longitudinally bissected fruit halves, oneeighth sections of fruit, one-quarter sections of fruit, whole carpel segments, 2 or 3 mm thick equatorial slices of fruit, and 1 cm2 fruit endocarp pieces) from 15 mm diam Citrus limon (L.) Burm. f. cv. Eureka lemons. Juice vesicles within equatorial fruit halves produced the least amount of callus. Furthermore, these juice vesicles grew similarly to juice vesicles occurring in the tree grown fruit. A study of cultured equatorial fruit halves using 10-45 mm diam lemons was then conducted. Fruit half cultures containing juice vesicles could be readily established from 15-45 mm diam lemons. Vesicles from 10 mm diam fruit halves, however, invariably produced callus. Vesicles cultured within fruit halves produced proportionately less callus as their fruit diam increased. Juice vesicles cultured in 15-30 mm diam fruits lost their original green color and turned opaque as they matured (i.e., after 3-6 months in culture). A method is also presented, whereby whole lemon fruits can be established and maintained in vitro. Lemons, 35-45 mm in diam, were the best explant sources for establishing whole fruit cultures. Juice vesicles in whole fruit cultures may remain viable for up to 8 months in culture. CHEMICAL TREATMENTS, environment, husbandry practices, genetic characters, and general plant health influence citrus fruit development and physiology (Sinclair and Bartholomew, 1944; Sauer, 1953; Reuther et al., 1969; Jahn and Young, 1972; El-Zeftawi, 1973; Boswell, Nauer, and Atkin, 1982). In the field, these factors are difficult to control and may obscure the influence of test environments, and of exogenous growth regulators and chemicals on citrus fruit development. Therefore, we sought to develop in vitro systems to study citrus fruit metabolism and growth in a controlled environment. Citrus fruit is a unique complex berry (also called a hesperidium) having 6-20 united carpels clustered around and joined to the floral axis to form locules into which seeds and juice sacs grow (Schneider, 1968). Phylogenetically, carpels are modified leaves. The economically important juice vesicles are unique to the subfamily Aurantioideae of the Rutaceae. Juice vesicles are appendages of the endocarp which ' Received for publication 25 January 1988; revision accepted 16 June 1988. Mention of a trademark, proprietary product or vendor does not constitute approval or guarantee of the product by the U.S. Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable. We would like to thank Drs. J. B. Carpenter and L. M. Blakely for critically reading the manuscript and Dr. M. Roose for providing plant material. through various morphogenetic and chemical stages give rise to the juice filled sacs. Over the last few decades several investigators have used various explants from citrus fruit to conduct growth and metabolism studies in vitro (Kordan, 1959, 1974, 1975; Murashige and Tucker, 1969; Schroeder, 1969; Chauhan and Roberts, 1978; Einset, 1978; Unger and Feng, 1978; Kato, 1980; Gulsen, Altman, and Goren, 1981; Altman, Gulsen, and Goren, 1982; Khan, Chauhan, and Roberts, 1986; Tisserat and Galletta, 1987). In these studies, citrus fruit tissue explants readily gave rise to callus. The production of callus from fruit tissue irreversibly alters both the normal morphogenetic and metabolic processes associated with fruit tissues. We report on the development of fruit culture systems to study juice vesicle growth where the preponderance of cultured vesicles remains callus-free (e.g., over 90%). Cultured juice vesicles, in our sterile fruit culture systems, developed similarly to vesicles within fruits grown on the tree. MATERIALS AND METHODS Fruits, 10-4 5 mm diam, of Citrus limon (L.) Burm. f. cv. Eureka (lemon) were obtained from 6-year old trees grown at the University of California, Riverside. Fruits were surface sterilized in a 2.63% sodium hypochlorite solution (containing 2 drops of Tween-20 emulsifier per 100 ml solution) for 30 min and then rinsed 3 times with sterile distilled water. The extreme stylar

Changes in activity of polyamine biosynthetic enzymes and in polyamine contents in developing fruit tissues of ‘Murcott’ mandarin

Arginine decarboxylase (ADC), ornithine decarboxylase (ODC), putrescine (Put) and spermidine (Spd) contents were analysed in growing ‘Murcott’ mandarin fruits from bloom to full development. Enzymes of polyamine synthesis and, to a lesser extent, polyamine contents roughly followed patterns of growth activity in fruit tissues, with a maximum at the early stages, a minimum at mid-summer and intermediate values at full fruit development. ADC/ODC and Put/Spd ratios were 2–8 and 2–30, respectively. Possible links between polyamine metabolism and citrus fruit set and development are suggested.

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Morphological studies on the development of citrus fruits. II.

Morphological studies on the development of citrus fruits. II.

The Story of Citrus Fruit Development

The Story of Citrus Fruit Development

Morphological studies on the development of citrus fruits I

Morphological studies on the fruit development of the Early Satsuma orange (Citrus unshiu MARC. var. praecox TANAKA), the Hassaku orange (C. Hassaku Y. TANAKA) and certain other species were made respectively. By comparing these results together with those obtained previously with the Satsuma orange, the general characteristics and specific or varietal differences in the citrus fruit growth were investigated. Noteworthy features of this investigation were as follows:1. In the growing citrus fruit, the peel and the pulp seem to show different growth cycles. At first, the peel increased in thickness rapidly, next came a vigorous growth of the pulp, which was again followed by a decrease in the peel thickness. Thereafter the peel and the pulp grew in harmony with one another.The peel continued to grow for a longer period of time than did the pulp. As the fruit became mature, the decline and the cessation of growth occurred earlier in the pulp than in the peel. It seems that the peel maintains its growth activity even after the fruit maturation, although the extent of this post-maturation activity varies among different forms. This characteristic of citrus was most clearly indicated by the conspicuous growth of the peel in the mature fruit of Shuto, a variety of C. aurantium LINN. during spring and summer.2. In young citrus fruit the peel increased its thickness rapidly when the cell multiplication in the albedo tissue became very vigorous. In the Hassaku orange, which has a thick peel, the albedo tissue cells multiplied more vigorously, continued to do so for a longer period, and as a result became greater in number than in the Satsuma orange.3. The development of citrus fruit was divided into three stages as follows: Stage I, the cell division period; Stage II, the cell enlargement period; and Stage III, the maturation period. The duration of each stage could be determined by external changes associated with the pulp growth. The period from the emergence of juice sacs to the beginning of their remarkable elongation was referred to as Stage I. In the Satsuma orange, it was observed previously that when the juice sacs began to enlarge abruptly most of their epidermal cells also initiated to elongate. This period was therefore referred to as the cell enlargement period, Stage II. Stage III was defined by a conspicuous decline of the growth in width of the pulp segments.The species and varieties of citrus fruits used in the present study differed in the durations of these three stages, whereby they could be arranged in the following order (from the longest to the shortest of each stage): Stage I-Hassaku orange, Early Satsuma orange, Satsuma orange; Stage II-Satsuma orange, Hassaku orange, Early Satsuma orange; Stage III- Hassaku orange, Satsuma orange, Early Satsuma orange. The fruit with longer duration of Stage I grew rapidly in Stage II and attained larger size. The duration of Stage II was not directly related either to the final fruit size or to the time of fruit maturity, while the latter was proportional to the duration of Stage III.4. Despite their larger size, the fruits of Hassaku and Iyokan (C. Iyo TANAKA) contained much fewer juice sacs than did those of the Early Satsuma orange and Satsuma oranges. Consequently, their juice sacs were relatively large, especially so in Hassaku. On the other hand, in every kind of citrus under observation there was a tendency within the same form that the larger the fruit the more juice sacs were found in it. However, the degree of this association was not uniform among different kinds. Thus there were greater regressions of the number of juice sacs on the pulp weight in the Early Satsuma orange or the Satsuma orange than in Hassaku or Iyokan.

Botanical-geographic orientation of citrus fruits with reference to their historical background

The progress of citrus taxonomy has been greatly delayed from various reasons, primarily due to the misapplication of specific names to classify obvious unit species. From the time of LINNAEUS, a num-ber of definite unit species of Citrus have been passes into two major groups symbolized by Citrus Medica and C. Aurantium, under which good unit species are placed in either one of them with the status of lower categories such as their subspecies, subvarieties and so on. HOOKER, and later ENGLER, followed the same erraneous scheme in using these specific names to group existing units which are quite independent from each other having definite characteristics with type materials, geographic dis-tribution and historical careers together with au-thentic descriptions and valid names worthy of bo-tanical species. C. Medica, for instance, is an unit species, representing the Turunj type of citron, known in India from the Sanskrit time under the name Matulunga, and in China from CHI-Han's period (A. D. 290_??_307) under Kou yuan. By simple resembrance, it cannot involve C. Limon (the lemon), C. Limonia (Canton lemon) and the lime (C. aurantifolia) of different taxonomic status and geographical independence. All of them are equally separate species of different origin and C. Medica cannot stand at an upper taxon to group them all. From this principle, every one of citrus units is to be handled equally to hold species rank so far as it represents valid specific status with definite geo-graphical background and historical bearing to prove its concrete existence. Higher taxa, such as sub-genus, section, subsection, etc., are to be created to systematize these unit species, which should not be in the shape of specific taxon, as these great authors unjustly misapplied against the present day type-concept. Minor difficulties blocking so far the smooth pathway of the taxonomic development of citrus fruits, are misidentification based upon graphical and historical absurdity, repeated abues of non-valid specific names, too frequent negligence of valid species by the lack of critical investi-gations, and the random use of obscure non-specific botanical units without proper justification. Citrus taxonomy, therefore, must proceed with the secure mapping of independent specific units authorized by taxonomic-geographical proof with historical endorsement if possible, then followed by their sys-tematic arrangement properly classified keeping the evolutionary sequence in sight.

Effect of Emanations from Several Species of Fungi on Respiration and Color Development of Citrus Fruits

Effect of Emanations from Several Species of Fungi on Respiration and Color Development of Citrus Fruits

Effect of Emanations from Several Species of Fungi on Respiration and Color Development of Citrus Fruits

Effect of Emanations from Several Species of Fungi on Respiration and Color Development of Citrus Fruits