GABA shunt pathway is stimulated in response to early defoliation-induced carbohydrate limitation in Mandarin fruits.

The regulation of sugar and organic acid metabolism during fruit development has a major effect on high-quality fruit production. The reduction of leaf area is a common feature in plant growth, induced by abiotic and biotic stresses and disturbing source/sink ratio, thus impacting fruit quality. Here, we induced carbohydrate limitation by partial leaf defoliation at the beginning of the second stage of mandarin development (before the citrate peak). Resulting changes were monitored in the short-term (48 h and 1 week) and long-term (7 weeks) after the defoliation. Short-term response to early defoliation implied metabolic settings to re-feed TCA for sustaining respiration rate. These features involved (i) vacuolar sucrose degradation (high acid invertase activity and mRNA expression level) and enhanced glycolytic flux (high ATP-phosphofructokinase activity), (ii) malic and citric acid utilization (increased phosphoenolpyruvate kinase and NADP-Isocitrate dehydrogenase) associated with vacuolar citric acid release (high mRNA expression of the transporter CsCit1) and (iii) stimulation of GABA shunt pathway (low GABA content and increased mRNA expression of succinate semialdehyde dehydrogenase). A steady-state proline level was found in ED fruits although an increase in P5CS mRNA expression level. These results contribute to a better knowledge of the molecular basis of the relationship between defoliation and sugar and organic acid metabolism in mandarin fruit.

Insight into Physiological and Biochemical Determinants of Salt Stress Tolerance in Tetraploid Citrus.

Citrus are classified as salt-sensitive crops. However, a large diversity has been observed regarding the trends of tolerance among citrus. In the present article, physiological and biochemical studies of salt stress tolerance were carried out according to the level of polyploidy of different citrus genotypes. We particularly investigated the impact of tetraploidy in trifoliate orange (Poncirus trifoliata (L.) Raf.) (PO4x) and Cleopatra mandarin (Citrus reshni Hort. Ex Tan.) (CL4x) on the tolerance to salt stress compared to their respective diploids (PO2x and CL2x). Physiological parameters such as gas exchange, ions contents in leaves and roots were analyzed. Roots and leaves samples were collected to measure polyphenol, malondialdehyde (MDA), ascorbate and H2O2 contents but also to measure the activities of enzymes involved in the detoxification of active oxygen species (ROS). Under control conditions, the interaction between genotype and ploidy allowed to discriminate different behavior in terms of photosynthetic and antioxidant capacities. These results were significantly altered when salt stress was applied when salt stress was applied. Contrary to the most sensitive genotype, that is to say the diploid trifoliate orange PO2x, PO4x was able to maintain photosynthetic activity under salt stress and had better antioxidant capacities. The same observation was made regarding the CL4x genotype known to be more tolerant to salt stress. Our results showed that tetraploidy may be a factor that could enhance salt stress tolerance in citrus.

Influence of Rootstock Genotype and Ploidy Level on Common Clementine (Citrus clementina Hort. ex Tan) Tolerance to Nutrient Deficiency.

Nutrient deficiency, in particular when this involves a major macronutrient (N, P, and K), is a limiting factor on the performance of plants in their natural habitat and agricultural environment. In the citrus industry, one of the eco-friendliest techniques for improving tolerance to biotic and abiotic stress is based on the grafting of a rootstock and a scion of economic interest. Scion tolerance may be improved by a tetraploid rootstock. The purpose of this study was to highlight if tolerance of a common clementine scion (C) (Citrus clementina Hort. ex Tan) to nutrient deficiency could be improved by several diploid (2×) and their tetraploid (4×) counterparts citrus genotypes commonly used as rootstocks: Trifoliate orange × Cleopatra mandarin (C/PMC2x and C/PMC4x), Carrizo citrange (C/CC2x and C/CC4x), Citrumelo 4475 (C/CM2x and C/CM4x). The allotetraploid FlhorAG1 (C/FL4x) was also included in the experimental design. The impact of nutrient deficiency on these seven scion/rootstock combinations was evaluated at root and leaf levels by investigating anatomical parameters, photosynthetic properties and oxidative and antioxidant metabolism. Nutrient deficiency affects foliar tissues, physiological parameters and oxidative metabolism in leaves and roots in different ways depending on the rootstock genotype and ploidy level. The best known nutrient deficiency-tolerant common clementine scions were grafted with the doubled diploid Citrumelo 4475 (C/CM4x) and the allotetraploid FlhorAG1 (C/FL4x). These combinations were found to have less foliar damage, fewer changes of photosynthetic processes [leaf net photosynthetic rate (P net ), stomatal conductance (g s ), transpiration (E), maximum quantum efficiency of PSII (F v /F m ), electron transport rate (ETR), ETR/P net ], and effective quantum yield of PSII [Y(II)], less malondialdehyde accumulation in leaves and better functional enzymatic and non-enzymatic antioxidant systems. Common clementine scions grafted on other 4× rootstocks did not show better tolerance than those grafted on their 2× counterparts. Chromosome doubling of rootstocks did not systematically improve the tolerance of the common clementine scion to nutrient deficiency.

Tetraploid Citrumelo 4475 rootstocks improve diploid common clementine tolerance to long-term nutrient deficiency.

Nutrient deficiency alters growth and the production of high-quality nutritious food. In Citrus crops, rootstock technologies have become a key tool for enhancing tolerance to abiotic stress. The use of doubled diploid rootstocks can improve adaptation to lower nutrient inputs. This study investigated leaf structure and ultrastructure and physiological and biochemical parameters of diploid common clementine scions (C) grafted on diploid (2x) and doubled diploid (4x) Carrizo citrange (C/CC2x and C/CC4x) and Citrumelo 4475 (C/CM2x and C/CM4x) rootstocks under optimal fertigation and after 7 months of nutrient deficiency. Rootstock ploidy level had no impact on structure but induced changes in the number and/or size of cells and some cell components of 2x common clementine leaves under optimal nutrition. Rootstock ploidy level did not modify gas exchanges in Carrizo citrange but induced a reduction in the leaf net photosynthetic rate in Citrumelo 4475. By assessing foliar damage, changes in photosynthetic processes and malondialdehyde accumulation, we found that C/CM4x were less affected by nutrient deficiency than the other scion/rootstock combinations. Their greater tolerance to nutrient deficiency was probably due to the better performance of the enzyme-based antioxidant system. Nutrient deficiency had similar impacts on C/CC2x and C/CC4x. Tolerance to nutrient deficiency can therefore be improved by rootstock polyploidy but remains dependent on the rootstock genotype.

Improved response of triploid citrus varieties to water deficit is related to anatomical and cytological properties.

Polyploidy plays a major role in citrus plant breeding to improve the adaptation of polyploid rootstocks as well as scions to adverse conditions and to enhance agronomic characteristics. In Citrus breeding programs, triploidy could be a useful tool to react to environmental issues and consumer demands because the produced fruits are seedless. In this study, we compared the physiological, biochemical, morphological, and ultrastructural responses to water deficit of triploid and diploid citrus varieties obtained from 'Fortune' mandarin and 'Ellendale' tangor hybridization. One diploid clementine tree was included and used as a reference. All studied scions were grafted on C-35 citrange rootstock. Triploidy decreased stomatal density and increased stomata size. The number of chloroplasts increased in 3x varieties. These cytological properties may explain the greater photosynthetic capacity (Pnet, gs, Fv/Fm) and enhanced water-holding capacity (RWC, proline). In addition, reduced degradation of ultrastructural organelles (chloroplasts and mitochondria) and thylakoids accompanied by less photosynthetic activity and low oxidative damages were found in 3x varieties. Triploid varieties, especially T40-3x, had a better ability to limit water loss and dissipate excess energy (NPQ) to protect photosystems. Higher starch reserves in 3x varieties suggest a better carbon and energy supply and increases in plastoglobuli size suggest less oxidative damage (H2O2, MDA), especially in T40-3x, and preservation of photosynthetic apparatus. Taken together, our results suggest that desirable cytological and ultrastructural traits induced by triploidy improve water stress response and could be a useful stress marker during environmental constraints.

Synthetic Polyploidy in Grafted Crops.

Synthetic polyploids have been extensively studied for breeding in the last decade. However, the use of such genotypes at the agronomical level is still limited. Polyploidization is known to modify certain plant phenotypes, while leaving most of the fundamental characteristics apparently untouched. For this reason, polyploid breeding can be very useful for improving specific traits of crop varieties, such as quality, yield, or environmental adaptation. Nevertheless, the mechanisms that underlie polyploidy-induced novelty remain poorly understood. Ploidy-induced phenotypes might also include some undesired effects that need to be considered. In the case of grafted or composite crops, benefits can be provided both by the rootstock's adaptation to the soil conditions and by the scion's excellent yield and quality. Thus, grafted crops provide an extraordinary opportunity to exploit artificial polyploidy, as the effects can be independently applied and explored at the root and/or scion level, increasing the chances of finding successful combinations. The use of synthetic tetraploid (4x) rootstocks may enhance adaptation to biotic and abiotic stresses in perennial crops such as apple or citrus. However, their use in commercial production is still very limited. Here, we will review the current and prospective use of artificial polyploidy for rootstock and scion improvement and the implications of their combination. The aim is to provide insight into the methods used to generate and select artificial polyploids and their limitations, the effects of polyploidy on crop phenotype (anatomy, function, quality, yield, and adaptation to stresses) and their potential agronomic relevance as scions or rootstocks in the context of climate change.

Nutrient Deficiency Tolerance in Citrus Is Dependent on Genotype or Ploidy Level.

Plants require essential minerals for their growth and development that are mainly acquired from soil by their roots. Nutrient deficiency is an environmental stress that can seriously affect fruit production and quality. In citrus crops, rootstock/scion combinations are frequently employed to enhance tolerance to various abiotic stresses. These tolerances can be improved in doubled diploid genotypes. The aim of this work was to compare the impact of nutrient deficiency on the physiological and biochemical response of diploid (2x) and doubled diploid (4x) citrus seedlings: Volkamer lemon, Trifoliate orange × Cleopatra mandarin hybrid, Carrizo citrange, Citrumelo 4475. Flhorag1 (Poncirus trifoliata + and willow leaf mandarin), an allotetraploid somatic hybrid, was also included in this study. Our results showed that depending on the genotype, macronutrient and micronutrient deficiency affected certain physiological traits and oxidative metabolism differently. Tetraploid genotypes, mainly Flhorag1 and Citrumelo 4475, appeared resistant compared to the other genotypes as indicated by the lesser decrease in photosynthetic parameters (P net, F v/F m, and G s) and the lower accumulation of oxidative markers (MDA and H2O2) in roots and leaves, especially after long-term nutrient deficiency. Their higher tolerance to nutrient deficiency could be explained by better activation of their antioxidant system. For the other genotypes, tetraploidization did not induce greater tolerance to nutrient deficiency.

Tetraploid citrus seedlings subjected to long-term nutrient deficiency are less affected at the ultrastructural, physiological and biochemical levels than diploid ones.

Nutrient deficiency has economic and ecological repercussions for citrus fruit crops worldwide. Citrus crops rely on fertilization to maintain good fruit output and quality, whereas new crop management policy aims to reduce fertilizers input. New rootstocks are needed to meet to this constraint, and the use of new tetraploid rootstocks better adapted to lower nutrient intake could offer a promising way forward. Here we compared physiological, biochemical and anatomic traits of leaves in diploid (2x) and doubled-diploid (4x) Citrumelo 4475 (Citrus paradisi L. Macf. × Poncirus trifoliata L. Raf.) and Volkamer lemon (Citrus limonia Osb.) seedlings over 7 months of nutrient deficiency. Photosynthetic parameters (Pnet, Gs and Fv/Fm) decreased, but to a lesser extent in 4x genotypes than 2x. Degradation of the ultrastructural organelles (chloroplasts and mitochondria) and compound cells (thylakoids and starches) was also lower in 4x genotypes, suggesting that tetraploidy may enhance tolerance to nutrient deficiency. However, leaf surface (stomata, stomatal density and epithelial cells) showed no nutrient deficiency-induced change. In 4x Citrumelo 4475, the higher tolerance to nutrient deficiency was associated with a lower MDA and H2O2 accumulation than in the 2x, suggesting a more efficient antioxidant system in the 4x genotype. However, few differences in antioxidant system and oxidative status were observed between 2x and 4x Volkamer lemons.

Tetraploid Carrizo citrange rootstock (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.) enhances natural chilling stress tolerance of common clementine (Citrus clementina Hort. ex Tan).

Low temperatures can disturb the development, growth and geographic distribution of plants, particularly cold-sensitive plants in the Mediterranean area, where temperatures can reach seasonally low levels. In citrus crops, scion/rootstock combinations are used to improve fruit production and quality, and increase tolerance to biotic and abiotic stresses. In the last decade, several studies have shown that tetraploid citrus seedlings or rootstocks are more tolerant to abiotic stress than their respective diploid. The objective of this study was to test whether the use of tetraploid rootstocks can improve the chilling tolerance of the scion. We compared physiological and biochemical responses to low seasonal temperatures of common Clementine (Citrus sinensis Osb.×Poncirus trifoliata L. Raf.) grafted on diploid and tetraploid Carrizo citrange rootstocks, named C/2xCC and C/4xCC, respectively. During the coldest months, C/4xCC showed a smaller decrease in net photosynthesis (Pn), stomatal conductance (Gs), chlorophyll fluorescence (Fv/Fm), and starch levels, and lower levels of malondialdehyde and electrolyte leakage than C/2xCC. Specific activities of catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) were higher in C/4xCC during the cold period, whereas chlorophyll, proline, ascorbate and hydrogen peroxide (H2O2) levels and superoxide dismutase (SOD) activity did not vary significantly between C/4xCC and C/2xCC throughout the study period. Taken together, these results demonstrate that tetraploid Carrizo citrange rootstock improves the chilling tolerance of common clementine (scion) thanks to a part of the antioxidant system.