Differential Expression of Organic Acid Degradation-Related Genes During Fruit Development of Navel Oranges (Citrus sinensis) in Two Habitats

Organic acids as well as soluble sugars contribute highly to flavor and overall quality of citrus fruit. Citric acid level in fruit is influenced by several factors including environmental conditions. In this study, it was observed that different environments in two habitats (Ganzhou, Jiangxi; Songyang, Zhejiang) had minor effects on total soluble solids and citrus color index but had significant effects on organic acids levels, particularly on citric acid level, in fruit of “Newhall” and “SkaggsBonanza” navel oranges (Citrus sinensis). Expression of genes involved in citric acid biosynthesis and degradation (CitCS1, CitCS2, CitAco1, CitAco2, CitAco3, CitIDH1, CitIDH2, CitIDH3, CitGAD4, CitGAD5, and CitGS2) was analyzed in fruit grown in each of the two habitats. Citric acid biosynthesis-related citrate synthase genes were steadily expressed during navel orange fruit development, while degradation-related genes were differentially expressed. These findings suggested that the influence of different environments on fruit quality traits was predominant on the regulation of organic acids level, particularly on the degradation of citric acid. A cascade of CitAco3–CitIDH1–CitGS2 might be involved in citric acid degradation in response to different environments during fruit growth and development.     

Involvement of CitCHX and CitDIC in Developmental-Related and Postharvest-Hot-Air Driven Citrate Degradation in Citrus Fruits

Citrate is the predominant organic acid associated with taste in citrus fruit. Although citrate metabolism has been widely studied in recent years, the potential contributions of transport proteins to citrate content remain unclear. In the present study, high-acid citrus fruit Gaocheng (‘GC’, Citrus sp.) and low-acid citrus fruit Satsuma mandarin (‘SM’, Citrus unshiu Marc.) were selected for study, and the degradation of citrate was deduced to be the main cause of the difference in acidity in fully mature fruits. RNA-seq analysis was carried out on ‘GC’ and ‘SM’ fruit samples over the same time course, and the results indicated that citrate degradation occurred mainly through the glutamine pathway, catalyzed by CitAco3-CitGS2-CitGDU1, and also two transport-related genes, CitCHX and CitDIC, were shown to be associated with citrate degradation. These results were confirmed by real-time PCR. In postharvest ‘GC’ fruit, the expressions of these two transport-related genes were induced by 2-fold under hot air treatment, accompanied by a reduction of 7%-9% in total acid degradation. Transient expression of CitCHX and CitDIC in tobacco leaves was performed, and the citrate content was reduced by 62%, 75% and 78% following CitCHX, CitDIC and CitCHX plus CitDIC treatments, respectively, as compared with expression of an empty vector. Overall, these data indicated that two transport proteins, CitCHX and CitDIC, are not only involved in citrate degradation during fruit development, but also involved in postharvest hot air triggered citrate reduction.     

Recent Advances in the Regulation of Citric Acid Metabolism in Citrus Fruit

The regulation of citric acid metabolism during fruit ripening has a major impact on the production of high-quality fruit. The impact of citric acid on organoleptic fruit quality attributes, fruit storage performance and the synthesis of several secondary metabolites has led to an exponential increase in research efforts during the last two decades. Recent research has focused on the relationship among citric acid biosynthesis, transportation, storage, and utilization. Among citrate metabolic processes, activities of a proton pump, especially the plasma membrane H⁺-ATPase on tonoplast and citrate catabolism in cytosol play important roles in the regulation of citrate accumulation in cell vacuoles. Moreover, we highlight recent advances and provide an overview of citrate metabolism, postharvest physiology of citrate metabolism, and the influence of agro-climatic factors on citrus fruits. It is the first review that provides a comprehensive model for citrate metabolism in citrus fruit juice sacs. We anticipate that this model for the regulation of citrate metabolism will facilitate the study of fruit acidity in citrus and other nonclimacteric fruits.     

The aconitate hydratase family from <Emphasis Type="Italic">Citrus</Emphasis>

Background  

Research on citrus fruit ripening has received considerable attention because of the importance of citrus fruits for the human diet. Organic acids are among the main determinants of taste and organoleptic quality of fruits and hence the control of fruit acidity loss has a strong economical relevance. In citrus, organic acids accumulate in the juice sac cells of developing fruits and are catabolized thereafter during ripening. Aconitase, that transforms citrate to isocitrate, is the first step of citric acid catabolism and a major component of the citrate utilization machinery. In this work, the citrus aconitase gene family was first characterized and a phylogenetic analysis was then carried out in order to understand the evolutionary history of this family in plants. Gene expression analyses of the citrus aconitase family were subsequently performed in several acidic and acidless genotypes to elucidate their involvement in acid homeostasis.     

Removal of RNA by heat treatment

Summary Eight bacterial strains were subjected to a discontinuous heat shock treatment aimed at causing a degradation of RNA. The treatment involved a 10 s to 10 min exposure to 65°C and then an incubation period of up to 3 h at 50°C. At intervals the cells were analyzed for RNA, DNA and protein. Whereas the contents of protein and DNA were not affected, RNA was degraded. An almost complete degradation of RNA occurred inAlcaligenes eutrophus H 16 — PHB^–4 andEscherichia coli K 12; only about 50% of the cellular RNA were degraded inPseudomonas putida andP.flava GA; inCorynebacterium autotrophicum 7 C,Nocardia opaca 1 b and coryneform strains 11 X and 30.1 b RNA degradation occurred only to a small extent.A continuous flow system for the treatment of cell suspensions by heat shock followed by incubation at an elevated temperature was developed. The results confirmed those obtained by batch-wise heat treatment.     

Induction and Citric Acid Productivity of Fluoroacetate-sensitive Mutant Strains of Candida lipolytica

To establish a novel process for the economical production of citric acid from n-paraffins by yeast, attempts were made to obtain some mutant strains capable of producing citric acid in higher yield without (+)-isocitric acid.

From among the mutant strains derived from Candida lipolytica ATCC 20114, which produced citric acid and (+)-isocitric acid in the ratio of about 60:40 from n-paraffins, a citrate non-utilizing mutant strain, K-20, and a fluoroacetate-sensitive mutant strain , S-22, were selected on the basis of high citric acid and low (+)-isocitric acid productivity.

The mutant strain S-22 showed extremely poor growth in a medium containing sodium citrate as the sole carbon source and extremely high sensitivity to fluoroacetate. The production ratio of citric acid and (+)-isocitric acid by the mutant strain was changed to 97:3, and the yield of the citric acid from n-paraffins, charged to the fermentation medium, reached 145%(w/w).