Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.     

Effects of zinc toxicity on sugar beet (Beta vulgaris L.) plants grown in hydroponics

The effects of high Zn concentration were investigated in sugar beet ( Beta vulgaris L.) plants grown in a controlled environment in hydroponics. High concentrations of Zn sulphate in the nutrient solution (50, 100 and 300 μ m ) decreased root and shoot fresh and dry mass, and increased root/shoot ratios, when compared to control conditions (1.2 μ m Zn). Plants grown with excess Zn had inward-rolled leaf edges and a damaged and brownish root system, with short lateral roots. High Zn decreased N, Mg, K and Mn concentrations in all plant parts, whereas P and Ca concentrations increased, but only in shoots. Leaves of plants treated with 50 and 100 μ m Zn developed symptoms of Fe deficiency, including decreases in Fe, chlorophyll and carotenoid concentrations, increases in carotenoid/chlorophyll and chlorophyll a / b ratios and de-epoxidation of violaxanthin cycle pigments. Plants grown with 300 μ m Zn had decreased photosystem II efficiency and further growth decreases but did not have leaf Fe deficiency symptoms. Leaf Zn concentrations of plants grown with excess Zn were high but fairly constant (230–260 μg·g⁻¹ dry weight), whereas total Zn uptake per plant decreased markedly with high Zn supply. These data indicate that sugar beet could be a good model to investigate Zn homeostasis mechanisms in plants, but is not an efficient species for Zn phytoremediation.     

Differential Responses of Iron,Magnesium, and Zinc Deficiency on Pigment Composition,Nutrient Content,and Photosynthetic Activity in Tropical Fruit Crops

Photosynthetica - Fe, Mn, and Zn affected the chlorophyll (Chl) content whereas Fe deficiency caused larger reduction of total chlorophyll content than Mg and Zn deficiencies. Mg deficient mango...     

Genetic analysis of the effect of zinc deficiency on Arabidopsis growth and mineral concentrations

Aims

Zinc deficiency is a common micronutrient deficiency in plants growing in many different regions of the world and is associated with disturbances in uptake and accumulation of mineral nutrients. Despite many published data on physiological factors affecting ion accumulation in Zn deficient plants, there is very little information about the genetic factors underlying this. We aim to identify genetic loci involved in mineral accumulation and plant performance under Zn deficiency.

Methods

Genetic loci were identified using the genetically segregating Ler × Cvi recombinant inbred line (RIL) population grown under Zn deficient conditions. Lines were analysed for the concentrations of Zn, Fe, Mn, K, Ca, Mg, P, Cu, S and Al in shoot dry matter. The same was done for the same lines grown under Zn sufficient conditions.

Results

We found considerable heritable variation for most mineral concentrations. In general, there was a positive correlation between mineral concentrations. For Zn only condition-dependent QTLs were identified, while for most other mineral concentrations both condition-dependent and -independent QTLs were identified. Several QTLs co-localize, including co-localization to loci controlling shoot biomass and to mineral concentration loci found previously in this and other RIL populations.

Conclusions

There are different genetic loci controlling Zn accumulation under deficient and sufficient Zn supply. Only for few minerals, their accumulation is controlled by Zn-supply-specific loci.     

Alfalfa genotypes differ in their ability to tolerate zinc deficiency

Response of 13 alfalfa (Medicago sativa L.) genotypes to varied Zn supply (+Zn: 2 mg kg⁻¹ soil, −Zn: no added Zn) was studied in a pot experiment under controlled environmental conditions. Plants were grown for four weeks in a Zn-deficient siliceous sandy soil. Plants grown at no added Zn showed typical Zn deficiency symptoms i.e. interveinal chlorosis of leaves, yellowish-white necrotic lesions on leaf blades, necrosis of leaf margins, smaller leaves and a marked reduction in growth. There was solute leakage from the leaves of Zn-deficient plants, while no solute leakage from Zn-sufficient plants. The ratios of P:Zn, Fe:Zn, Cu:Zn and Mn:Zn in Zn-deficient plants were extremely high compared with Zn-sufficient plants indicating disturbance of P:Zn, Fe:Zn, Cu:Zn and Mn:Zn balance within plant system by Zn deficiency. Genotypes differed markedly in Zn efficiency based on shoot dry matter production. Alfalfa genotypes also differed markedly in P:Zn ratio, Cu:Zn ratio and Fe:Zn ratio under —Zn treatment. The shoot dry weight, shoot:root ratio, chlorophyll content of fresh leaf tissue, solute leakage from the leaves, Zn uptake and distribution of Zn in shoots and roots were the most sensitive parameters of Zn efficiency. Zn-efficient genotypes had less solute leakage but higher shoot:root ratio and higher Zn uptake compared with Zn-inefficient genotypes. Under —Zn treatment, Zn-inefficient genotypes had less Zn partitioning to shoots (33–37%) and more Zn retained in roots (63–67%), while Zn-efficient genotypes had about equal proportions of Zn in roots (50%) and shoots (50%). This revised version was published online in June 2006 with corrections to the Cover Date.     

An analysis of the growth response of carrot seedlings to deficiency in some mineral nutrients

Critical plant concentrations for a reduction in relative growth rate to 90% of that of fully nourished plants were estimated by a novel method for several mineral nutrients. Carrot plants were grown from seed for 28 days in a range of nutrient solutions omitting N, P, K, Ca, S, Mg, Fe, B, Mn, Zn, Cu and Mo as separate treatments. All treatments except -Mn, -Zn, -Cu and -Mo resulted in effects on plant growth and the development of deficiency symptoms. Estimates of critical concentrations were based on a simple simulation model incorporating the principle of nutrient dilution with increasing plant weight and on mineral analysis of the plants. Parameters governing the shape of the relationship between fractional relative growth rate and plant nutrient concentration were altered until the model predicted the observed final mean dry weight of deficient plants and time of divergence of this growth curve from that of fully nourished plants. Critical concentrations so obtained were higher than those previously reported for Ca, Fe, N and P in carrots and lower for K, Mg and S.     

Induction of the Root Cell Plasma Membrane Ferric Reductase (An Exclusive Role for Fe and Cu)

Induction of ferric reductase activity in dicots and nongrass monocots is a well-recognized response to Fe deficiency. Recent evidence has shown that Cu deficiency also induces plasma membrane Fe reduction. In this study we investigated whether other nutrient deficiencies could also induce ferric reductase activity in roots of pea (Pisum sativum L. cv Sparkle) seedlings. Of the nutrient deficiencies tested (K, Mg, Ca, Mn, Zn, Fe, and Cu), only Cu and Fe deficiencies elicited a response. Cu deficiency induced an activity intermediate between Fe-deficient and control plant activities. To ascertain whether the same reductase is induced by Fe and Cu deficiency, concentration- and pH-dependent kinetics of root ferric reduction were compared in plants grown under control, -Fe, and -Cu conditions. Additionally, rhizosphere acidification, another process induced by Fe deficiency, was quantified in pea seedlings grown under the three regimes. Control, Fe-deficient, and Cu-deficient plants exhibited no major differences in pH optima or Km for the kinetics of ferric reduction. However, the Vmax for ferric reduction was dramatically influenced by plant nutrient status, increasing 16- to 38-fold under Fe deficiency and 1.5- to 4-fold under Cu deficiency, compared with that of control plants. These results are consistent with a model in which varying amounts of the same enzyme are deployed on the plasma membrane in response to plant Fe or Cu status. Rhizosphere acidification rates in the Cu-deficient plants were similarly intermediate between those of the control and Fe-deficient plants. These results suggest that Cu deficiency induces the same responses induced by Fe deficiency in peas.     

Latent manganese deficiency in barley can be diagnosed and remediated on the basis of chlorophyll a fluorescence measurements

Background and aims

Manganese (Mn) deficiency represents a major plant nutritional disorder in winter cereals. The deficiency frequently occurs latently and the lack of visual symptoms prevents timely remediation and cause significant yield reductions. These problems prompted us to investigate chlorophyll (Chl) a fluorescence as a tool for diagnosis of latent Mn deficiency.

Methods

Barley plants grown under controlled greenhouse conditions or in the field were exposed to different intensities of Mn deficiency. The responses were characterised by analysis of Chl a fluorescence, photosystem II (PSII) proteins and mineral elements.

Results

Analysis of the Chl a fluorescence induction kinetics (FIK) revealed distinct changes long before any visual symptoms of Mn deficiency were apparent. The changes were specific for Mn and did not occur in Mg, S, Fe or Cu deficient plants. The changes in Mn deficient plants were accompanied by a marked reduction of the D1 protein in PSII. Foliar Mn application fully restored PSII functionality, ensured winter survival, and increased grain yields under field conditions.

Conclusions

The efficiency and stability of PSII are markedly affected by latent Mn deficiency. Chlorophyll a fluorescence measurements constitute a powerful and valuable tool for diagnosis and remediation of latent Mn deficiency.     

Zinc deficiency as a critical problem in wheat production in Central Anatolia

In a soil and plant survey, and in field and greenhouse experiments the nutritional status of wheat plants was evaluated for Zn, Fe, Mn and Cu in Central Anatolia, a semi-arid region and the major wheat growing area of Turkey.All 76 soils sampled in Central Anatolia were highly alkaline with an average pH of 7. 9. More than 90% of soils contained less than 0.5 mg kg^-1 DTPA-extractable Zn, which is widely considered to be the critical deficiency concentration of Zn for plants grown on calcareous soils. About 25% of soils contained less than 2.5 mg kg^-1 DTPA-extractable Fe which is considered to be the critical deficiency concentration of Fe for plants. The concentrations of DTPA-extractable Mn and Cu were in the sufficiency range. Also the Zn concentrations in leaves were very low. More than 80% of the 136 leaf samples contained less than 10 mg Zn kg^–1. By contrast, concentrations of Fe, Mn and Cu in leaves were in the sufficient range.In the field experiments at six locations, application of 23 kg Zn ha^-1 increased grain yield in all locations. Relative increases in grain yield resulting from Zn application ranged between 5% to 554% with a mean of 43%. Significant increases in grain yield (more than 31%) as a result of Zn application were found for the locations where soils contained less than 0.15 mg kg^-1 DTPA-extractable Zn.In pot experirnents with two bread (Triticum aestivum, cvs. Gerek-79 and Kirac-66) and two durum wheats (Triticum durum, cvs. Kiziltan-91 and Kunduru-1149), an application of 10 mg Zn kg^-1 soil enhanced shoot dry matter production by about 3.5-fold in soils containing 0.11 mg kg^-1 and 0.15 mg kg^-1 DTPA-extractable Zn. Results from both field observations and greenhouse experiments showed that durum wheats were more susceptible to Zn deficiency than the bread wheats. On Zn deficient soils, durum wheats as compared to bread wheats developed deficiency symptoms in shoots earlier and to a greater extent, and had lower Zn concentration in shoot tissue and lower Zn content per shoot than the bread wheats.The results presented in this paper demonstrate that (i) Zn deficiency is a critical nutritional problem in Central Anatolia substantially limiting wheat production, (ii) durum wheats possess higher sensitivity to Zn deficient conditions than bread wheats, and (iii) wheat plants grown in calcareous soils containing less than 0.2 mg kg^-1 DTPA-extractable Zn significantly respond to soil Zn applications. The results also indicate that low levels of Zn in soils and plant materials (i.e. grains) could be a major contributing factor for widespread occurrence of Zn deficiency in children in Turkey, whose diets are dominated by cereal-based foods.     

Structural and functional integrity of Sulla carnosa photosynthetic apparatus under iron deficiency conditions

• The abundance of calcareous soils makes bicarbonate‐induced iron (Fe) deficiency a major problem for plant growth and crop yield. Therefore, Fe‐efficient plants may constitute a solution for use on calcareous soils.
• We investigated the ability of the forage legume Sulla carnosa (Desf.) to maintain integrity of its photosynthetic apparatus under Fe deficiency conditions. Three treatments were applied: control, direct Fe deficiency and bicarbonate‐induced Fe deficiency.
• At harvest, all organs of deficient plants showed severe growth inhibition, the effect being less pronounced under indirect Fe deficiency. Pigment analysis of fully expanded leaves revealed a reduction in concentrations of chlorophyll a, chlorophyll b and carotenoids under Fe deficiency. Electron transport rate, maximum and effective quantum yield of photosystem II (PSII), photochemical quenching (qP), non‐photochemical quenching (qN) as well as P700 activity also decreased significantly in plants exposed to direct Fe deficiency, while qN was not affected. The effects of indirect Fe deficiency on the same parameters were less pronounced in bicarbonate‐treated plants. The relative abundances of thylakoid proteins related to PSI (PsaA, Lhca1, Lhca2) and PSII (PsbA, Lhcb1) were also more affected under direct than indirect Fe deficiency.
• We conclude that S. carnosa can maintain the integrity of its photosynthetic apparatus under bicarbonate‐induced Fe deficiency, preventing harmful effects to both photosystems under direct Fe deficiency. This suggests a high capacity of this species not only to take up Fe in the presence of bicarbonate (HCO₃^?) but also to preferentially translocate absorbed Fe towards leaves and prevent its inactivation.

     

Interactive effects of salinity and iron deficiency in Medicago ciliaris

In calcareous salt-affected soils, iron availability to plants is subjected to the effects of both sodium and bicarbonate ions. Our aim was to study interactive effects of salinity and iron deficiency on iron acquisition and root acidification induced by iron deficiency in Medicago ciliaris L., a species commonly found in saline ecosystems. Four treatments were used: C, control treatment, complete medium (CM) containing 30 microM Fe; S, salt treatment, CM with 75 mM NaCl; D, deficient treatment, CM containing only 1 microM Fe; DS, interactive treatment, CM containing 1 microM Fe with 75 mM NaCl. Our study showed that plant growth and chlorophyll content were much more affected by the interactive treatment than by iron deficiency or by the salt treatment, indicating an additive effect of these constraints in DS plants. These results could be partially explained by Na accumulation in shoots as well as a limitation of nutrient uptake such as Fe and K under salt stress, under iron deficiency, and especially under their combined effect. The study also showed that root acidification was deeply diminished when iron deficiency was associated with salinity. This probably explained the decrease of Fe uptake and suggested that root proton pump activity would be inhibited by salinity.     

Mechanism of phosphorus-induced zinc deficiency in cotton. I. Zinc deficiency-enhanced uptake rate of phosphorus

The effect of withholding Zn on the uptake, translocation and accumulation of P was studied in cotton plants ( Gossypium hirsutum L. cv. Deltapine 15/21) grown in nutrient solutions under controlled environmental conditions. The influence of P on the uptake rate, translocation and distribution of ⁶⁵Zn in the plants was also examined. Increasing the P supply resulted in severe Zn deficiency symptoms (interveinal chlorosis) as well as P toxicity symptoms, which were characterized by leaf puckering and grayish-brown marginal necrosis. Zinc deficiency markedly increased the uptake and translocation rates of P over the whole concentration range tested (5x10^-5 to 1.25x10^-3 M ). Uptake and translocation rates of P increased with both level of P and severity of Zn deficiency. This often caused P toxicity symptoms on Zn-deficient leaves. In contrast to P, the concentrations of K and Mg in the leaves were not affected by Zn deficiency. Similar results were obtained for sunflower ( Helianthus annuus L.) and buckwheat ( Fagopyrum esculentum Moench) plants. Higher P concentrations in Zn-deficient leaves or shoots could not be attributed wholly to reduced shoot growth. This was also evident when Zn deficiency was compared with other micronutrient (Fe, Mn, and Cu) deficiencies. Only Zn-deficient plants showed enhanced uptake and translocation of P. In experiments with ⁶⁵Zn, a high P supply did not depress uptake and translocation of Zn. From the results obtained it is concluded that the P-induced Zn deficiency in cotton, as well as in other species, is primarily caused by enhanced P uptake and translocation and not by inhibition of Zn uptake.     

Response of soybean genotypes to boron,zinc and manganese deficiency in tissue culture

The effect of boron (B), zinc (Zn), and manganese (Mn) deficiency in the medium on eight soybean genotypes was evaluated using friable callus after a 21 day growth period. Of the three micronutrients evaluated, Zn and B deficiencies had the greatest effect on callus weight, while Mn had only a slight effect. Despite this, significant differences in callus weight reduction of the eight genotypes tested were observed on the three different media. These results indicate that genotypic variation for response to B, Zn, and Mn deficiency is present in soybean at the cellular level. If the resistant genotypes identified in this study are also resistant at the whole plant level, then they could be used in soils which are deficient in the above micronutrients.     

Differential tolerance to iron deficiency of chickpea varieties and Fe resupply effects

The consequences of direct iron deficiency and iron resupply were evaluated during development stages of two Tunisian chickpea varieties (INRAT88 and Chetoui) cultivated in continuously aerated solution with or without 20 muM Fe. The chlorosis score was estimated during culture. Growth parameters, chlorophyll concentration, acidification capacity and Fe concentration were measured every three days during the 21-day treatment. After three weeks of treatment, the chlorosis index was 3-fold higher in Chetoui than in INRAT88, and a considerable decrease of chlorophyll concentration was observed in Chetoui plants since the 6th day of -Fe deprivation. Iron deficiency significantly inhibited whole-plant biomass deposition in both varieties. However, the growth reduction appeared earlier, and was more pronounced in Chetoui than in INRAT88. The whole-plant Fe content decreased dramatically under deficient conditions, and we note an Fe enrichment in shoots at the expense of roots. The sensitivity of Chetoui as compared to INRAT88 was confirmed by the behaviour of resupplied (-Fe/+Fe) plants. In fact, the addition of iron to deficient plants had no significant effect in Chetoui, whereas it led to a total recovery in INRAT88. The capacity of INRAT88 to maintain plant growth and to preserve adequate chlorophyll synthesis under limited iron conditions is related to its better Fe-use efficiency, in addition to its capacity to rapidly recover from this stress.     

Effect of zinc nutritional status on activities of superoxide radical and hydrogen peroxide scavenging enzymes in bean leaves

The effect of varied zinc (Zn) supply on the activities of superoxide dismutase (SOD), ascorbate (AsA) peroxidase, glutathione (GSSG) reductase, catalase and guaiacol peroxidase was studied in leaves of bean (Phaseolus vulgaris) plants grown for 15 days in nutrient solution. Zinc deficiency severely decreased plant growth and the leaf concentrations of soluble protein and chlorophyll. Resupply of Zn to deficient plants for up to 72h restored protein concentrations more rapidly than chlorophyll and plant growth. With the exception of guaiacol peroxidase, the activities of all enzymes were significantly decreased by Zn deficiency, in particular GSSG reductase and SOD. Within 72h of resupplying Zn to deficient plants, the enzyme activities reached the level of the Zn sufficient plants. The results indicate severe impairment in the ability of Zn-deficient leaves to enzymically scavenge O₂ ^- and H₂O₂. Consequences and reasons of this impairment are discussed in terms of photooxidation of chloroplast pigments and inhibition of the biosynthesis of the related scavenger enzyme proteins.     

铁蛋白基因表达对烟草耐低铁能力的影响

铁是植物生长发育的必需元素。由于土壤中的三价铁离子不能被植物直接利用, 使一些植物经常表现出缺铁症状。为探讨利用铁蛋白基因提高植物耐低铁胁迫的作用, 利用农杆菌介导法将大豆铁蛋白基因SoyFer1和内源反义铁蛋白基因NtFer2的cDNA分别导入烟草基因组, 采集转基因烟草种子。对T1转基因烟草的卡那霉素抗性分析表明, 整合到烟草基因组的外源基因多为单拷贝基因, 也有少数为多拷贝基因。对具有卡那霉素抗性的转基因植株进行PCR检测和Northern杂交分析表明, 外源基因已整合到烟草基因组中, 并且得到了正确表达。将转基因株系移栽到铁离子浓度不同的培养基中生长2个月后进行比较表明, 转大豆铁蛋白基因烟草株系的生长量明显高于非转基因烟草株系, 而转内源反义铁蛋白基因烟草株系的生长量则明显低于非转基因烟草株系。转大豆铁蛋白基因和转内源反义铁蛋白基因烟草株系的叶绿素含量、丙二醛(MDA)含量和过氧化物酶(POD)活性等生理性状也发生了明显变化, 表现为转大豆铁蛋白基因株系的叶绿素含量明显增加, POD活性明显增强, MDA含量明显降低; 而转内源反义铁蛋白基因株系的叶绿素含量、POD活性和MDA含量等则表现为与转大豆铁蛋白基因株系的相反。铁蛋白过量表达提高了烟草耐低铁能力, 而铁蛋白抑制表达则降低了烟草耐低铁能力。     

Arbuscular mycorrhizas modify plant responses to soil zinc addition

Zinc deficiency is one of the most commonly reported plant and human nutrient deficiencies worldwide. Conversely, Zn is also a common environmental contaminant, significantly reducing plant growth. These contrasting effects of Zn on plant growth and nutrition have been the focus of a considerable number of studies; however, most studies focus on plant responses to soil Zn concentration under either deficient or toxic concentrations, but not both. The formation of arbuscular mycorrhizas (AM) can increase plant Zn uptake under low soil Zn concentrations, and on the other hand, ‘protect’ plants against excessive Zn accumulation under high soil Zn conditions. Here we report the findings of an experiment in which we studied the response of AM formed by tomatoes under low, medium and high soil Zn conditions. To control for the formation of AM in this study we used a mycorrhiza defective tomato mutant and its mycorrhizal wildtype progenitor. While mycorrhizal colonization was not significantly impacted by soil Zn addition, the growth of plants and tissue Zn concentrations were. Together these data highlight the complex interactions between AM and Zn, and the utility of a genotypic approach for studying AM in this context.     

Effects of phosphorus and iron fertilizers on the growth of two soybean varieties at two soil temperatures

The influence of FeEDDHA (0, 0.2 and 2 μg Fe g⁻¹ soil) and NaH₂PO₄·H₂O (0 and 120 μg Pg⁻¹ soil) on the growth of two Fe-ineffective soybean (Glycine max L. Merr.) varieties (anoka and T203) on a calcareous soil at two soil temperatures (16 and 24°C) was compared under greenhouse conditions. The two soybean varieties differed in the following respects: (a) T203 accumulated smaller concentrations of Fe in washed tops than Anoka under comparable conditions; (b) T203 was more susceptible to Fe deficiency and its accentuation by high levels of fertilizer P than Anoka; (c) T203 accumulated lower quantities of Mn in tops than Anoka under comparable conditions; (d) T203, but not Anoka, developed Mn deficiency symptoms when treated with P and 2 μg Fe g⁻¹ at 16°C. Fe deficiency was more severe in both varieties at the higher soil temperature due apparently to: (a) greater plant concentration of P in tops at 24°C; and/or (b) an increased rate of plant growth and greater dilution of Fe in young tissue at 24°C. Foliar P concentration was increased much more than foliar Fe concentration by an increase in soil temperature. Severely Fe deficient T203 plants grown without FeEDDHA at 24°C accumulated less foliar Mn than their FeEDDHA counterparts. Comparisons of Fe effectiveness of various soybean cultivars based on relative responses to FeEDDHA can be influenced by differential effects on Mn nutrition.     

Carboxylating enzymes and carbonic anhydrase functions were suppressed by zinc deficiency in maize and chickpea plants

Maize and chickpea plants were grown in a controlled environment with 0.5 M Zn or without Zn and various photosynthetic reactions were studied. The chlorophyll level, the rate of photosynthesis and photosystem II activity, the activity of carboxylating enzymes and that of carbonic anhydrase were suppressed by Zn deficiency in both plant species. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was quantified using polyacrylamide gel electrophoresis. Growing plants in a medium without Zn caused a decrease in the total protein level and in the levels of large and small subunits of Rubisco.