Visual effects of excluding EDTA from the growth medium
In early experiments, we observed negative effects on seedlings and rosette plants when omitting the EDTA chelator from the growth medium, and the effects were strikingly stronger for lcmt1 as compared with WT (Fig. 1). After germination of seedlings on complete MS medium, seedlings were transferred to fresh MS medium or MS medium lacking EDTA. This resulted in chlorosis of shoots and impairment of root growth (Fig. 1). Apparently lcmt1 was less resistant to the stress exerted by lack of EDTA, with strongly inhibited root growth (Fig. 1b). To explore effects of stressful conditions on a later developmental stage, plants were cultivated in soil for three weeks, then moved to rock wool with Hoagland solution with or without EDTA and containing FeCl3 or FeSO4 as iron source (Fig. 1c-f). Both WT and lcmt1 grew well with Hoagland prepared with EDTA in combination with FeCl3 or FeSO4 (Fig. 1c, e). When plants were given FeCl3 without EDTA both WT and lcmt1 became chlorotic (Fig. 1d), and growth of lcmt1 was strongly hampered. When plants were given FeSO4 as iron source without a chelator (Fig. 1f), also both WT and lcmt1 became chlorotic, and the stress symptoms were again more severe for lcmt1 (Fig. 1d). Clearly, as for seedlings, omitting EDTA stressed the plants and lcmt1was more affected than WT.
Iron-EDTA
To further get insight into different stress responses of
lcmt1 seedlings, the concentration of Fe-EDTA (FeNa
2EDTA) was varied from 0 to 500 mM in the agar medium (Fig. 2, Supplemental Fig. S1). Optimal seedling growth was observed at 25 and 50 mM (concentrations in half-strength and regular MS medium) for both WT and
lcmt1. Shoots of
lcmt1 generally had lower fresh weight than WT. At standard concentration (25 mM Fe-EDTA) fresh weight of
lcmt1 shoots was 70% of WT fresh weight. To be able to compare changes in
lcmt1 and WT related to the different treatments, all data was standardized to fresh weight and root elongation at 25 mM set to one (Fig. 2). After growth for six days with different iron concentrations, shoot weight of WT seedlings was lower by approximately 45 and 70% when exposed to 350 and 500 mM Fe-EDTA, respectively, but no significant difference in decreases was seen for
lcmt1 as compared with WT. However, from 200 mM and higher Fe-EDTA there was a negative effect on root elongation in
lcmt1 as compared with WT. At 500 mM, roots of both WT and
lcmt1 stopped growing (Supplemental Fig. S1). The results showed that high concentrations of Fe-EDTA impaired root growth in
lcmt1 more strongly than in WT (Fig. 2b). Interestingly, at zero Fe-EDTA root growth was also more strongly affected in
lcmt1 than in WT.
Zinc and other micronutrients
The effects of excluding EDTA from the agar medium in the presence and absence of various micronutrients were tested and showed that fresh weight of shoots was severely reduced in response to lack of EDTA or lack of all micronutrients (Fe, Zn, B, Mn, Mo, Cu, Co, I) (Fig. 3). Addition of 16 mM zinc (standard concentration) led to increased fresh weight, but only when comparing media containing EDTA. A ten times higher, 160 mM, concentration of zinc did not have positive effects. In conclusion, when avoiding the micronutrients, fresh weight of both WT and lcmt1 was more than 50% reduced, but there were no obvious differences between the responses of lcmt1 and WT regarding fresh weight (Fig 3a). On the other hand, growth of roots was more strongly impaired in lcmt1 compared with WT in all media lacking micronutrients and/or EDTA (Fig. 3b).
Aluminium
Shoot fresh weight of seedlings grown on agar decreased when Al2(SO4)3 was added in concentrations of 0.4 or 0.6 mM, but with no significant difference in response between WT and lcmt1 (Fig. 4a). Roots of lcmt1 grew significantly less in the presence at concentrations 0.2, 0.4, and 0.6 mM as compared with WT (Fig. 4b, Fig. S2.).
Heat stress
Seedlings were exposed to 37oC for 6-48 h and compared with non-exposed controls. After 18 h or longer time at 37oC the seedlings gained less shoot fresh weight during the subsequent 6 days, but there was no difference between WT and lcmt1 (Fig. 5a, Fig. S3). Negative effects on root growth were observed after 12 h of heat stress (Fig. 5b). After 24, 36 and 48 h, the effects on root growth were stronger for lcmt1 than for WT (Fig. 5b, Fig. S3).
Heat stress was also tested with rosette plants grown in soil for 5 weeks. Plants were exposed to heat (37oC) for 0, 8, 18, 24 h, then grown at 22oC. Pictures were taken i) immediately after treatment, ii) after 1 week and iii) after 2 weeks (Fig.6, Fig. S4). Both WT and lcmt1 grew well after 8 or 18 h heat treatment (Fig S4). After 24 h heat treatment, inspecting the plants after 1 week of recovery showed more green leaves for WT in three repeats (Fig. 6, Fig. S4), however, after the second week all plants died indicating that the shoot meristem was impaired in both lcmt1 and WT.
ROS are common metabolic intermediates and a result of many different types of stress, including metal stress and heat stress. Experiments confirmed that also ROS, in the form of H2O2 added to the agar medium at 0.5-2.5 mM), resulted in stronger inhibition of root growth in lcmt1 than in WT (Supplemental Fig S5).
Element analysis
Element analysis was performed for shoots of seedlings grown in agar in Petri dishes without or with EDTA for 12 days, and for shoots of 6-week-old rosette stage plants grown first on soil for three weeks, then on rock wool for 3 weeks without or with EDTA, or without and with iron. When comparing seedlings of lcmt1 and WT, the differences in element contents were generally less than 10% or too variable to become significant (Table 1 and supplemental Table S1). When comparing seedlings grown without or with EDTA (pooling WT and lcmt1) there was a clear difference for zinc content; seedlings grown without EDTA having 48% more zinc than seedlings with EDTA. For iron and sulphur, when omitting EDTA, there was a 12% decrease or increase, respectively (Table 1).
For 6-week-old plants on rock wool, as for seedlings, significant differences between element contents in rosettes of lcmt1 and WT were generally not found, but with one interesting exception; sodium content was elevated by 64% in lcmt1. The elevated sodium level for lcmt1 persisted also for plants grown without iron, on soil, or with extra aluminium (Supplemental Table S1). As for seedlings, when grown without EDTA, rosette plants had increased levels of zinc (increased by 89%) (Table 1). For the rosette stage plants, other micronutrients (Cu, Mn, Mo, Ni) also showed elevated levels in the absence of EDTA. From the elevated elements, especially Cu, Mn and Zn were present at high concentrations 40, 185 and 59 mg/g DW, respectively (data based on Table S1). Such high metal levels may have influenced several metabolic reactions and led to impaired growth and chlorosis (Fig. 1d, f). Other micronutrients responding significantly when omitting EDTA were molybdenum and nickel, but these represented only 8 and 3 mg/g DW, and would hardly be harmful. Apparently, in the absence and presence of EDTA, homeostasis mechanisms kept iron constant (Table 1, Table S1), but growth was impaired. When iron was excluded from the nutrient solution (but EDTA was kept), the level of iron in rosettes decreased by 41% (Table 1, bottom row). Altogether the element analysis showed clear effects of excluding the chelator in the nutrient solution, especially by increased levels of the micronutrients Cu, Mn and Zn, however, significant differences related to the lcmt1 mutation was found only for sodium, with increased levels in lcmt1 plants.
Table 1.Relativeelement contents in shoots of seedlings on agar or shoots of plants on rock wool. Seedlings: seeds were sown in Petri dishes, grown for 12 days in MS medium without or with EDTA. Rosettes: plants were first grown for 3 weeks in soil, then transferred to rock wool for 3 more weeks with different nutrient solutions. The Hoagland solution was made without or with EDTA, or without and with iron. Data are given for i) an element in lcmt1 seedlings as percentage of that element in WT seedlings; ii) an element in seedlings grown in the absence of EDTA as percentage of that element in seedlings grown in the presence of EDTA; iii) an element in lcmt1 rosettes as percentage of that element in WT rosettes; iv) an element in rosettes grown in the absence of EDTA as percentage of that element in rosettes grown in the presence of EDTA; v) an element in rosettes grown in the absence of iron as percentage of that element in rosettes grown with iron. P values are given for all comparisons. Percentages that were significantly different from 100 with p< 0.05 by TTEST are high-lighted in red,
RNAseq
After sowing and three days at 4oC, WT and lcmt1 seedlings were germinated and grown under stressful conditions in Petri dishes by omitting EDTA from the growth medium during 5 days at 22oC, then harvesting. WT and lcmt1 seedlings still looked very similar and were considered suitable for comparison of changes in gene expression caused by lack of EDTA. Some seedlings were left to grow for another week and confirmed the strong growth inhibition on lcmt1 when EDTA was omitted for some more days (as in Fig. 1b). Almost 3 000 genes showed significantly different expression levels in lcmt1 and WT, 1528 genes were up-regulated and 1366 genes were down-regulated in lcmt1 in comparison with WT. When the limit was set to 2x different expression levels, 520 genes were up-regulated and 653 genes were down-regulated in lcmt1 as compared with WT. Several genes (155) were more than 5x downregulated in lcmt1 (Table 2, Supplemental Table S2 with original data).
Table 2. Number of genes differentially expressed in lcmt1 and
WT when grown on ½ MS medium lacking EDTA.
|
Number of genes
|
Genes significant differently expressed
|
2894
|
Genes up-regulated in lcmt1
|
1528
|
Genes down-regulated in lcmt1
|
1366
|
|
|
Genes > 5x different in lcmt1 and WT
|
194
|
Genes > 5x up-regulated in lcmt1
|
39
|
Genes > 5x down-regulated in lcmt1
|
155
|
|
|
Genes > 2x different in lcmt and WT
|
1173
|
Genes > 2x up-regulated in lcmt1
|
520
|
Genes > 2x down-regulated in lcmt1
|
653
|
GO (Gene Ontology) enrichment was analysed by help of Panther (Mi et al., 2021) which divides genes into groups belonging to three categories; Biological processes, Molecular function, and Cellular components. Examples of enriched ontologies for genes 2x differently expressed are presented in Table 3. Many enriched groups extensively overlapped, therefore, not all groups are listed. Complete lists are presented in the Supplemental material (Supplemental Tables S3-S8). Genes up-regulated in lcmt1 were enriched in iron homeostasis genes, synthesis of cutin and various stress-annotated genes. Iron homeostasis genes were further inspected (Fig. 7). These genes included five BHLH transcription factors and the BRUTUS E3 ligase involved in regulation of iron homeostasis regulation, two genes directly involved in uptake of iron from the environment i.e. IRT1 and FRO2, the vacuolar localized metal transporters FRD3 and NRAMP4, and the oligopeptide transporter gene encoding a phloem specific iron transporter gene OPT3. We manually added three IMA genes (IRONMAN1,2,6). Other IMA genes, IMA 3 and 4 were also 4-5 times higher expressed in lcmt1, but are not included in the figure. IMA genes were recently shown to be crucial for regulation of iron transport and homeostasis (Grillet, Lan, Li, Mokkapati, & Schmidt, 2018). We also added genes involved in chelating or storage of iron. Three NAS genes (1, 2, 4) encoding enzymes that synthesise the chelator nicotianamine were upregulated, whereas the iron storage protein FER1 was downregulated in lcmt1, indicating less storage capacity. The PP2AA3 gene, used as a control, was constant. Many stress related genes were differentially expressed in lcmt1 and WT, they were either up (97) or down (128) regulated in lcmt1. Transporter genes were also differentially expressed, both up (41) and down regulated (128) in lcmt1 (Table 3). Genes involved in photosynthesis, i.e. light harvesting, were strikingly downregulated in lcmt1. Also, genes encoding hem-binding proteins many of which are cytochromes, were down-regulated in lcmt1. The ZINC-INDUCED FACILITATOR (ZIF1) was expressed at a high level and 2.9 times higher in lcmt1 than in WT seedlings (Table S2). ZIF1 is important for transportation of nicotianamine and Zn into the vacuole and critical for both Zn and Fe homeostasis (Haydon et al., 2012). The heat shock protein HSP90-1 known to be involved in many different types of stresses was 2.2 times higher in lcmt1 than WT (Table S2).
Table 3. GO (Gene ontology) enrichment. Statistical over/under representation (powered by Panther, https://www.arabidopsis.org/tools/go_term_enrichment.jsp).Genes expressed at a level at least 2 times higher or lower in lcmt1 as compared with WT were included in the analysis (Details are shown in Table S3-S8).
|
number
of genes
|
fold enriched
|
Biological processes. 2x higher in lcmt1
|
|
|
|
iron ion homeostasis
|
10
|
12.80
|
|
cutin biosynthesis
|
5
|
20.62
|
|
response to water deprivation
|
18
|
3.72
|
|
response to osmotic stress
|
30
|
4.22
|
|
response to temperature stimulus
|
25
|
2.97
|
|
response to stress
|
97
|
3.31
|
|
|
|
|
Molecular function. 2x higher in lcmt1
|
|
|
|
transmembrane transporter activity
|
41
|
2.47
|
Cellular component. 2x higher in lcmt1
|
|
none
|
Biological processes. 2x lower in lcmt1
|
|
|
|
Photosynthesis, light harvesting in photosystem 1
|
8
|
20.30
|
|
Response to light stimulus
|
43
|
3.68
|
|
Defence response to other organisms
|
58
|
4.38
|
|
Response to stress
|
128
|
2.50
|
|
Response to ethylene
|
13
|
5.07
|
|
Response to salicylic acid
|
19
|
7.66
|
|
Response to oxygen levels
|
16
|
3.66
|
|
Response to temperature
|
30
|
2.92
|
|
Response to fungus
|
27
|
4.70
|
|
Response to bacteria
|
43
|
5.11
|
|
|
|
Molecular function. 2x lower in lcmt1
|
|
|
|
Anion transmembrane transporter activity
|
24
|
2.72
|
|
Chlorophyll binding
|
7
|
15.22
|
|
Heme binding
|
23
|
3.76
|
|
oxidoreductase activity
|
49
|
2.12
|
Cellular component. 2x lower in lcmt1
|
|
|
|
plastoglobule
|
9
|
7.21
|