Proteomic analysis of Citrus sinensis roots and leaves in response to long-term magnesium-deficiency

Background

Magnesium (Mg)-deficiency is frequently observed in Citrus plantations and is responsible for the loss of productivity and poor fruit quality. Knowledge on the effects of Mg-deficiency on upstream targets is scarce. Seedlings of ‘Xuegan’ [Citrus sinensis (L.) Osbeck] were irrigated with Mg-deficient (0 mM MgSO₄) or Mg-sufficient (1 mM MgSO₄) nutrient solution for 16 weeks. Thereafter, we first investigated the proteomic responses of C. sinensis roots and leaves to Mg-deficiency using two-dimensional electrophoresis (2-DE) in order to (a) enrich our understanding of the molecular mechanisms of plants to deal with Mg-deficiency and (b) understand the molecular mechanisms by which Mg-deficiency lead to a decrease in photosynthesis.

Results

Fifty-nine upregulated and 31 downregulated protein spots were isolated in Mg-deficient leaves, while only 19 upregulated and 12 downregulated protein spots in Mg-deficient roots. Many Mg-deficiency-responsive proteins were involved in carbohydrate and energy metabolism, followed by protein metabolism, stress responses, nucleic acid metabolism, cell wall and cytoskeleton metabolism, lipid metabolism and cell transport. The larger changes in leaf proteome versus root one in response to Mg-deficiency was further supported by our observation that total soluble protein concentration was decreased by Mg-deficiency in leaves, but unaffected in roots. Mg-deficiency had decreased levels of proteins [i.e. ribulose-1,5-bisphosphate carboxylase (Rubisco), rubisco activase, oxygen evolving enhancer protein 1, photosynthetic electron transfer-like protein, ferredoxin-NADP reductase (FNR), aldolase] involved in photosynthesis, thus decreasing leaf photosynthesis. To cope with Mg-deficiency, C. sinensis leaves and roots might respond adaptively to Mg-deficiency through: improving leaf respiration and lowering root respiration, but increasing (decreasing) the levels of proteins related to ATP synthase in roots (leaves); enhancing the levels of proteins involved in reactive oxygen species (ROS) scavenging and other stress-responsive proteins; accelerating proteolytic cleavage of proteins by proteases, protein transport and amino acid metabolism; and upregulating the levels of proteins involved in cell wall and cytoskeleton metabolism.

Conclusions

Our results demonstrated that proteomics were more affected by long-term Mg-deficiency in leaves than in roots, and that the adaptive responses differed between roots and leaves when exposed to long-term Mg-deficiency. Mg-deficiency decreased the levels of many proteins involved in photosynthesis, thus decreasing leaf photosynthesis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1462-z) contains supplementary material, which is available to authorized users.     

Persistence of [14C] gibberellin A3 and [3H] gibberellin A1 in senescing,ethylene treated Citrus and tomato fruit

The fate of [¹⁴C] gibberellin A₃ and [³H] gibberellin A₁ was examined in senescing fruit of Shamouti orange (Citrus sinensis L. Osbeck) and tomato (Lycopersicon esculentum Mill.). Gibberellin A₃ was highly persistent in Citrus peel (t 1/2=18 days) and to a lesser degree in tomato (t 1/2=5.5 days). Ethylene and ethephon caused a slight enhancement of gibberellin A₃ metabolism in Citrus and tomato fruit, respectively. Gibberellin A₁ was metabolized by Citrus peel at a relatively high rate (t 1/2 < 24 h) and ethylene slightly reduced this rate. It is concluded that the ethylene-induced enhancement of senescence does not involve major effects on the deactivation of applied gibberellins.Abbreviations GA₃ gibberellin A₃ - GA₁ gibberellin A₁     

Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis

Tetraploid plants are essential for interploid hybridization to create triploid seedless citrus. Here we report a simple and efficient in vitro method for generating autotetraploids for sweet orange (Citrus sinensis). Cell-division activity in ‘Anliucheng’ sweet orange callus was analyzed using flow cytometry to determine the peak frequency of cell division at which time callus in a liquid media and solid media was treated with 1000 mg l⁻¹ colchicine. The percentage of the DNA-content-varied cells in the callus increased markedly from 11.0% to 44.4% and to 59.0% for liquid and solid media respectively. A total of 20 tetraploid plantlets were recovered via embryogenesis from 47 plantlets regenerated from the treated callus. All the autotetraploids were derived from different embryoids. Autotetraploids will be useful parents for interploid hybridization to generate commercially valuable seedless triploid citrus cultivars.     

Proteomic analysis of somatic embryogenesis in Valencia sweet orange (Citrus sinensis Osbeck)

Two dimensional gel electrophoresis combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to study the somatic embryogenesis (SE) in Valencia sweet orange (Citrus sinensis Osbeck). Twenty-four differentially expressed proteins were identified at five time points of citrus SE (0, 1, 2, 3, 4 weeks after embryo initiation) covering globular, heart/torpedo and cotyledon-shaped embryo stages. The general expression patterns for these proteins were consistent with those appeared at 4 weeks of citrus SE. The most striking feature of our study was that five proteins were predicted to be involved in glutathione (GSH) metabolism and anti-oxidative stress, and they exhibited different expression patterns during SE. Based on that oxidative stress has been validated to enhance SE, the preferential representation for anti-oxidative proteins suggests that they could have a developmental role in citrus SE. Some proteins involved in cell division, photosynthesis and detoxification were also identified, and their possible roles in citrus SE were discussed.     

Uniformity of plants regenerated from orange (Citrus sinensis Osb.) protoplasts

Summary Using 25 plants (protoclones) regenerated from orange (Citrus sinensis Osb.) protoplasts, several characters, including leaf and flower morphology, leaf oil, isozyme patterns and chromosome number, were examined. No significant variations in each character were recorded among the protoclones. Uniformity observed among protoclones was identical to that of nucellar seedlings.     

Plant regeneration from undeveloped ovules and embryogenic calli of Citrus: Embryo production,germination, and plant survival

Embryogenic cultures were initiated from undeveloped ovules of several polyembryonic Citrus species on a basal medium supplemented with either malt extract, 2,4-D alone, or 2,4-D in combination with BA or daminozide. Primary embryos of all responsive cultivars were harvested directly from ovule cultures; secondary embryo harvests were made from Handin orange ovule cultures and long-term embryogenic callus. Differences were observed among cultivars and treatments in percentage of responsive ovules and total number of embryos produced. The most effective treatment for embryo production varied among cultivars. Embryo germination and plant establishment frequencies were determined for this plant regeneration system. Differences among cultivars with respect to regenerate survival percentage were minimal. Plant regeneration via secondary or long-term callus-derived embryos was as efficient as from primary embryos. Critical factors influencing plant production and survival were the production of normal viable embryos, balanced germination, and successful acclimatization to the external environment.     

Identification and Expression Pattern of a Novel NAM,ATAF, and CUC-Like Gene from <Emphasis Type="Italic">Citrus sinensis</Emphasis> Osbeck

A citrus NAM, ATAF, and CUC (NAC)-like gene (CitNAC) was isolated from fruit tissues of Citrus sinensis Osbeck using complementary DNA (cDNA) amplified fragment length polymorphism and rapid amplification of cDNA ends techniques. Its full length was 988 bp in which 781 bp form the open reading frame, coding for a protein of 264 amino acids. Sequence comparison revealed that CitNAC possesses the general structural features at the N terminus of the NAC domains. Phylogenetic analysis results showed that CitNAC was closely related to AtNAP and PeNAP, which are involved in plant organ senescence. Gene expression analysis showed that the messenger RNA level of CitNAC was just detected in fruit peel and pulp during fruit ripening or senescence stage. The observed expression pattern of CitNAC along with the result of phylogenetic analysis suggested that CitNAC is related to fruit development and senescence.     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

Abscisic acid and indole-3-acetic acid contents in orange trees infected by Xylella fastidiosa and submitted to cycles of water stress

Pêra sweet orange plants (Citrus sinensis L. Osbeck) grafted on Rangpur lime rootstock (1 year-old) (Citrus limonia Osbeck) were inoculated with Xylella fastidiosa, a xylem-limited bacterium pathogen, which causes Citrus Variegated Chlorosis (CVC). Since it was known that water deficiency in the field enhances CVC-effects on the plant, the trees were submitted to three cycles of water stress during a one year period (March and October, 1998; and April, 1999) and divided in four treatments: healthy plants (HP); water-stressed healthy plants (WSHP); diseased plants (DP) and water-stressed diseased plants (WSDP). Stomatal conductance (g _s) of water-stressed diseased plants decreased in the first and second cycles of water deficiency, as the stress was increasing. The low stomatal conductance verified may be due to the high concentrations of abscisic acid (ABA) found in these plants. In the third cycle, values of g _s in diseased plants were, usually, lower than in the healthy ones. In healthy plants, g _s was reduced when these plants were submitted to water deficiency, independently of the cycle. The drop in leaf water potential in healthy plants was faster after irrigation was withheld, because healthy plants transpired more and, therefore, the water content of the substrate decreased more quickly. When the irrigation of WSDP was withheld in the third cycle, it was not possible to detect increases in ABA contents, suggesting that other factors could be acting to diminish the stomatal conductance in these plants. The presence of Xylella fastidiosa did not induce an increase in indole-3-acetic acid content in the leaves. After three cycles of water deficiency, the concentrations of indole-3-acetic acid in WSHP and WSDP were lower than those concentrations in the irrigated controls on the day water stress was more severe.     

Growth and development of Citrus pistils and fruit explants in vitro

Pistils and various fruit explants of Citrus limon L. Burm. f. and Citrus sinensis L. Osbeck were cultivated in vitro . Basal medium as well as medium supplemented with IAA, GA₃ or benzylaminopurine, supported growth of all explants for more than one year. Pistils did not enlarge considerably, but gave rise to active callus growth; callus proliferation and viability was enhanced by all hormones. Culture of fruit slice explants resulted, in addition to peel hypertrophy and callus proliferation, in a marked growth of two distinct types of juice vesicles. The growth of juice vesicle explants was promoted by all three growth hormones. – It is suggested that the successfully prolonged in vitro culture of various fruit explants, and especially of juice vesicles, may aid in studies of fruit development and physiology.     

Plant regeneration of sweet orange (Citrus sinensis) from thin sections of mature stem segments

An efficient system for in vitro plant regeneration from thin transversal stem sections explants (1–2 mm) using mature tissues of sweet orange cv. Pera was developed. Explants were cultured in different media to evaluate the frequency of regeneration and size of buds. A high percentage of explants (54% with 3.1 buds/explant) producing large buds (1–4 mm) was observed when the explants were cultivated for 2 weeks on Murashige and Skoog medium and then transferred to Woody Plant medium (WPM). Both media were supplemented with 1.8 M 6-benzylaminopurine and 0.7 M gibberellic acid. Adventitious buds were regenerated into whole plants by in vitro shoot-tip grafting. Regenerated plants started to flower after 12 months in the greenhouse, confirming their mature nature.     

Clonal variation of tea [Camellia sinensis (L.) O. Kuntze] in countering water deficiency

Various clones of tea [Camellia sinensis (L.) O. Kuntze] such as TTL-1, TTL-2, TTL-4, TTL-5, TTL-6, UPASI-2 and UPASI-3 planted in the field were subjected to soil moisture stress conditions by withholding irrigation. A control set of the same clones were maintained by watering regularly. The soil water content of the irrigated and non irrigated plants was monitored through the soil moisture status. The extent of effect of drought on tea plants were monitored through various physiological parameters such as shoot weight, leaf water potential, chlorophyll and carotenoid content, chlorophyll fluorescence (Fv/Fm), net photosynthetic rate, transpiration rate, stomatal conductance and biochemical parameters such as extent of proline accumulation and free radical generation. These parameters were studied on the 30 d of non irrigation and on the 5 d during recovery from drought. The plants recovered when re-irrigated after 30 d of non-irrigation, which suggests that permanent wilting did not occur due to non-irrigation up to 30 d. On the 30 d of non-irrigation the clones TTL-1, TTL-6 and UPASI-2 showed lesser reduction of shoot weight, leaf water potential, chlorophyll fluorescence, photosynthetic rate, transpiration rate and stomatal conductance and increased proline and lesser lipid peroxidation as compared to the other clones. From these results it can be concluded that the clones TTL-1, TTL-6 and UPASI-2 are comparatively more drought tolerant than the clones TTL-2, TTL-4, TTL-5 and UPASI-3.     

Expression of a senescence-associated cysteine protease gene related to peel pitting of navel orange (Citrus sinensis L. Osbeck)

Previously, a suppression subtractive hybridization library was constructed to identify differentially expressed genes in peel pitting of navel orange fruit and a cDNA fragment sharing high similarities to cysteine protease genes was identified. In this study, we cloned its full-length cDNA sequence, designated CsCP, using the Rapid amplification of cDNA ends approach. It consists of 1,409 nucleotides and its ORF encodes 361 amino acids predicted to have an N-terminal signal peptide. Phylogenetic analysis revealed that CsCP belonged to the aleurain group in papain family of cysteine proteases. According to quantitative RT-PCR, the expression of CsCP was enhanced during the development of postharvest peel pitting concomitant with senescence, although it was detectable in all tested tissues including root, leaf, flower and peel of fruit. RNA gel blot analysis showed that the CsCP expression was induced by hypoxia (3% O₂), but repressed by anoxia (0% O₂), wounding, ethylene and high temperature (40°C). Conclusively, the CsCP is a senescence-associated gene and up-regulated during the development of citrus postharvest peel pitting, which provides a basis to understand its role in citrus peel pitting.     

Photosynthesis and transpiration of healthy and diseased spruce trees in the course of three vegetation periods

Summary CO₂- and H₂O-gas exchange of 20- to 25-year-old spruce trees from a plantation in the Hunsrück mountains were investigated over a period of 3 years. All measurements were made as pair comparisons, i.e., in each case the gas exchange of a damaged tree and of a relatively healthy tree in its immediate vicinity was measured simultaneously. A second plantation in the Westerwald mountains consisted of 18-year-old apparently healthy spruce trees. Pair comparison at this location meant comparison of two healthylooking trees. The investigations at both locations included diurnal course measurements of photosynthesis and transpiration, and light saturation curves and CO₂-saturation curves of photosynthesis. The reduced photosynthesis parameters of the phenotypically damaged trees at the Hunsrück location indicates massive damage to the photosynthetic apparatus. Measurements of H₂O-gas exchange showed that there are disturbances in stomatal regulation of the needles of damaged trees. As a result, the water use efficiency of these needles proved to be significantly lower. In addition, apparent photorespiration of the damaged trees was decreased, whereas their light- and CO₂-compensation points and their dark respiration were increased. In contrast to the Hunsrück plantation, no such effects were detectable when the healthy-looking Westerwald trees were subjected to pair comparison of gas exchange. Reduced photosynthetic capacity and disturbances of the stomatal regulation of the phenotypically damaged Hunsrück trees may be due to damage in the cellular membranes. Furthermore, a comparison of three growing seasons led to the conclusion that the gas exchange of spruce trees in their natural habitat is markedly influenced by climatic conditions.     

Effect of Salt Stress on the Superoxide Dismutase Activity in Leaves of Citrus limonum in Different Rootstock-Scion Combinations

The effect of salinity on leaf superoxide dismutase (SOD) activity of lemon trees of different rootstock-scion combinations was studied. In leaves from Citrus limonum cv.Verna scions on Citrus macrophylla and C. reticulata rootstocks, salinity treatment clearly caused a significant depression in both Fe-SOD and Mn-SOD activities and an increase in Cu,Zn-SOD activity. However, in leaves from Citrus limonum on Citrus aurantium rootstook, the reduction observed in the activity values of Fe-SODs and Mn-SODs was not statistically significant. Salt stress also produced a decrease in the content of soluble proteins and chlorophylls. However, this drop was greater in C. limonum leaves on C. macrophylla than for other combinations.     

Transpiration-induced changes in the photosynthetic capacity of leaves

High transpiration rates were found to affect the photosynthetic capacity of Xanthium strumarium L. leaves in a manner analagous to that of low soil water potential. The effect was also looked for and found in Gossypium hirsutum L., Agathis robusta (C. Moore ex Muell.) Bailey, Eucalyptus microcarpa Maiden, Larrea divaricata Cav., the wilty flacca tomato mutant (Lycopersicon esculentum (L.) Mill.) and Scrophularia desertorum (Munz) Shaw. Two methods were used to distinguish between effects on stomatal conductance, which can lower assimilation by reducing CO₂ availability, and effects on the photosynthetic capacity of the mesophyll. First, the response of assimilation to intercellular CO₂ pressure (C _i) was compared under conditions of high and low transpiration. Second, in addition to estimating C _i using the usual Ohm's law analogy, C _i was measured directly using the closed-loop technique of T.D. Sharkey, K. Imai, G.D. Farquhar and I.R. Cowan (1982, Plant Physiol, 60, 657–659). Transpiration stress responses of Xanthium strumarium were compared with soil drought effects. Both stresses reduced photosynthesis at high C _i but not at low C _i; transpiration stress increased the quantum requirement of photosynthesis. Transpiration stress could be induced in small sections of leaves. Total transpiration from the plant did not influence the photosynthetic capacity of a leaf kept under constant conditions, indicating that water deficits develop over small areas within the leaf. The effect of high transpiration on photosynthesis was reversed approximately half-way by returning the plants to low-transpiration conditions. This reversal occurred as fast as measurements could be made (5 min), but little further recovery was observed in subsequent hours.Abbreviations and symbols A photosynthetic CO₂ assimilation rate - C _a ambient CO₂ partial pressure - C _i partial pressure of CO₂ inside the leaf - VPD leaf-to-air water-vapor pressure difference This research was begun while the author was a Postdoctoral Research Fellow at the Australian National University, Canberra     

Photosynthesis and water relations and the role of anatomy in Umbilicariaceae (lichenes) from Central Spain

Summary The response of net photosynthesis and dark respiration in eight species of Umbilicariaceae (lichenes) to temperature (-5, 0, 5, 10, 15, 20, 25, 30°C) and irradiance (55, 110, 220, 400, 620 mol photons m^-2 s^-1 PAR) was studied. The samples were collected in montane and alpine localities of the Spanish Sistema Central. The species differed widely in their net photosynthetic rates. The optimal temperature for net photosynthesis in alpine species was significantly lower than in montane species. Montane species were more photophytic than alpine ones. Water saturation and water loss rate were dependent on morphology and particularly anatomy of the thallus. The physiological and structural data are useful in the interpretation of the ecology and altitudinal distribution of the Umbilicariaceae. No adaptation could be linked to particularities of the mediterranean climate.     

3种藓类植物水分含量与光合作用、呼吸作用和水势的关系

对湿地匍灯藓〔Plagiomniumacutum(Lindb.)T.Kop.〕、大羽藓〔Thuidiumcymbifolium(Dozy&Molk.)Dozy&Molk.〕和垂藓〔Chrysocladiunretrorsum(Mitt.)Fleisch.〕的水分含量与光合作用、呼吸作用和水势的关系进行了初步研究（1999年5月20日到6月10日）。在这3种藓类植物中,其水分含量与光合作用速率（Pn）的关系可以分为2种类型一种类型如大羽藓和垂藓,在藓体水分含量20%～70%时,Pn随着水分含量增加而增加,但是在80%～95%时,Pn随水分含量增加而下降,光合最适水分含量约70%～80%;另一种出现在湿地匍灯藓,水分含量20%～80%时,Pn随着水分含量增加而增加,在80%～95%时,Pn维持一个较高的水平,光合最适水分含量为80%～90%。在一个大的水分含量范围内（60%～95%）,暗呼吸（Rd）保持相对稳定,但是在水分含量较低时（20%～70%）,Rd随着水分含量的下降而下降。在藓体水分含量与水势之间的关系方面,3种藓类植物相似,水分含量与水势对数之间的回归曲线为S形曲线。