1. Recent research has found Sedum alfredii capable of subverting the toxic effects of high copper concentrations based on highly efficient hyperaccumulator molecular mechanisms found in proteins known
as phytochelatins and metallothioneins. The questioned posed within this research is whether these molecular mechanisms are common to the Sedum species such as Sedum reflexum. As copper's toxic effects cause decreased function in
photosynthetic electron transport, oxidative stress, and subsequent growth inhibition, chl-a concentration and visual growth analysis was studied. Additionally, any hyperaccumulation mechanisms would sequester copper within the root
systems in order to prevent access to sensitive systems; therefore, biomass and ICP-MS analysis was used in order to confirm copper sequestration within the root systems. Based on two way ANOVA and post-hoc Tukey tests, statistical
differences were found over time and concentration for relative chl-a levels. A relative decrease in 6 μM to 24 μM in chl-a concentration when compared to the control indicates that the copper was having a negative impact on
the Sedum reflexum plants. Visual plant growth analysis, when compared with the control, displayed increased whitening along the stem, shortened stems, and leaf curling. Plants subjected to increased copper concentrations displayed root
structures which were more bundled than linear, but the decreased lengths changed from day to day. The biomass analysis showed statistical differences over time only specifically 48 hours after the copper concentration was added, but the
relative trend does indicate increased biomass in all copper concentrations after Day 6 when compared with the control. Any further analysis for copper retention would need to be confirmed by ICP-MS analysis. Conclusively, over the 22
day period, there were no cases of plant senescence due to copper concentration. Any plants that were analyzed continued to display new leaf formations, and while their chl-a concentrations levels were decreased, there was no complete loss
of function. This indicates, based on supplemental studies performed on Arabidopsis Thaliana, that Sedum reflexum may contain some increased hyperaccumulator mechanisms compared to model organisms, but further studies would need
to be performed in order to determine whether the mechanisms are as efficient as Sedum alfredii.
Copper is an essential redox-active transition metal capable of accepting and donating up to two
electrons based on availability of its outer orbital shell. This mechanism assists in cellular growth
and development, but due to its highly reactive nature it is heavily regulated. As a trace element,
the sufficiency level is between 5-20 μg/g or copper concentrations between 0.5 μM to 6 μM. At
toxic levels the consequences include growth inhibition, DNA damage and oxidative stress.
Plant mechanisms localize copper away from sensitive sites such as photosynthesis. Some
examples include movement restriction to roots by ectomycorrhiza, vacuole sequestering and
chelation in the cytosol by phytochelatins and metallothioneins. In addition, these proteins assist in
repair and protection of the plasma membrane and transport excess metals to vacuoles. When an
organism is capable of highly efficient upregulation of these proteins, they are known
as hyperaccumulators.
In China, Sedum alfredii was found near mining sites and is capable of retaining more than
500 μg/g of copper. The Sedum species as succulents have unique features that allow them to
handle extreme environments with little water due to storage organs and CAM mechanisms. Their
photosynthetic features are mainly within their stems rather than leaves. The question posed is
whether another Sedum species, Sedum reflexum, also contains hyperaccumulator mechanisms or
if the succulent's other unique qualities may be the cause for surviving under high levels of
toxicity. Physical manifestations common to copper toxicity such as leaf curling, decreased
biomass, decreased relative chl-a concentrations, would need to be analyzed in order to determine
whether Sedum reflexum is capable of handling copper levels beyond 6 μM.
The main purpose of this research is to analyze any changes to plant growth, photosynthetic
structures, and biomass increase due to copper retention that may occur to Sedum reflexum under
high copper concentration. Sedum reflexum photosynthetic structures are apparent at
approximately 6 weeks old and the growth rate of 0.5 g/𝑚2
per day helped to determine any visible
change in plant growth over time.
The methodology is as follows:
Germination occurred for approximately two weeks in petri dishes at 25C with 5ml of RO water
added every two days.
Establishment occurred between 2 to 4 weeks after first leaflet set was visually seen.
Plants were placed in starter pot kits individually with Vigoro Organics organic soil mixture.
Additional Copper was added during Day 1, 3, 9 and 15 at 6 μM, 12 μM and 24 μM.
Tests that were used to analyze the plants with three replications.
Visual Plant growth: Pictures were taken during every day of analysis to see if there were any
structural changes
Relative Chl-a concentrations: live plant mass was macerated with mortar and pestle then re-
suspended in 90% acetone. The relative Chl-concentrations are based on the live plant mass
weight, 10 ml sample and chl-a concentration obtained from TD-700 Turner Design Fluorometer
Biomass: Plants were dried for two hours and then samples were weighed.
ICP-MS analysis: Any samples weighed from the Biomass analysis were then oven dried at 495 C,
and re-suspended in 1N HCl and RO water.
After 22 days, 2-way ANOVA with replication was performed followed by a post-hoc Tukey test.
Based on two way ANOVA and post-hoc Tukey test, Based on two way ANOVA and post-hoc Tukey test, statistical differences were found over time and concentration for relative chl-a
levels. A relative decrease in 6 μM to 24 μM in chl-a concentration when compared to the control indicates that the copper was having a negative impact on the Sedum Reflexum plants. This
impact was found on days directly after copper concentration was added. Despite the control's growth over a 4 week span, the relative chl-a concentrations were consistently between 150 –
200 μg/g. What this indicates is that the decreases pertaining to the subsequent copper subjected plants, which reached levels as low as 50 μg/g, were due to the copper, and there was no
known compensation over time due to growth. The changes in chl-a concentration could be seen based on the visual plant growth analysis, where the pigmentation decreased along the
stem, an area where high levels of photosynthesis occur in succulents. Further speculation can be made that the copper was reaching the stem locations and inhibiting photosynthetic
electron transport. Once certain areas were inhibited, the electron excitation obtained from chlorophyll would be unable to pass through the photosystems and would subsequently damage
the chlorophyll in the process known as leaf chorosis. Biomass analysis showed statistical differences over time specifically 48 hours after the copper concentration was added. A trend was
seen over time showing an increase in biomass from plants subjected to copper, but further analysis would need to be confirmed by ICP-MS in order to determine whether this is due to
copper sequestering. Conclusively, over the 22 day period, no plant senescence occured despite the copper concentration being between two to four times higher than the
sufficiency. Studies performed on Arabidopsis Thaliana displayed significant changes in root systems as high as a 25% decrease in biomass at only 5 μM. This suggests
that Sedum Reflexum may contain similar efficient hyperaccumulator molecular mechanisms as Sedum Alfredii. Further studies would need to be performed to analyze how much copper
can be retained by Sedum Reflexum beyond 24 μM as relative chl-a concentrations were already decreasing.
Conclusions
ResultsIntroduction
Methodology
St Bonaventure University 2014 Effects of excess copper levels on Sedum reflexum seedlings
Growth and Photosynthetic Structures
By Christy A Twilight A.S., Dr. Theodore Georgian
References
• Cobbett, C., & Goldsbrough, P. (2002). Phytochelatins
and metallothioneins: roles in heavy metal detoxification
and homeostasis. Annual review of plant biology, 53(1),
159-182.
• Durhman, A. K., Rowe, D. B., & Rugh, C. L. (2006).
Effect of watering regimen on chlorophyll fluorescence
and growth of selected green roof plant
taxa. HortScience, 41(7), 1623-1628.
• Küpper, H. Compartmentation and complexation of
metals in hyperaccumulator plants. Frontiers in Plant
Science, 0
• Lequeux, H., Hermans, C., Lutts, S., & Verbruggen, N.
(2010). Response to copper excess in Arabidopsis
thaliana: Impact on the root system architecture, hormone
distribution, lignin accumulation and mineral
profile. Plant Physiology and Biochemistry, 48(8), 673-
682.
• Peñarrubia, L. (2013). Comparison of global responses to
mild deficiency and excess copper levels in Arabidopsis
seedlings. Metallomics, 5(9), 1234-1246.
• Yruela, Inmaculada. (2005). Copper in plants. Brazilian
Journal of Plant Physiology, 17(1), 145-156. Retrieved
March 20, 2014, from
http://www.scielo.br/scielo.php?script=sci_arttext&pid=S
1677-04202005000100012&lng=en&tlng=en.
10.1590/S1677-04202005000100012.
0
50
100
150
200
250
300
350
Day 0 Day 2 Day 5 Day 6 Day 12 Day 13 Day 22
AverageRelativeChl-aConcentration(ug/g)
Time
T 0 uM
T 6 uM
T 12 uM
T 24 uM
*
**
*/**
+/* P < 0.01
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
Day 0 Day 2 Day 5 Day 6 Day 12 Day 13 Day 22
AverageWeight(g)
Time
T 0 uM
T 6 uM
T 12 uM
T 24 uM
*
** **
P < 0.01
Figure 1. Average Biomass of Sedum reflexum plants under additional copper
concentrations from 0 uM to 24 uM over time. Tukey indicated differences at Day
6 and Day 13 approximately 48 hours after copper was added. Day 13 was different
from all other days. After Day 6, from respect to 0 uM, there is a increase in
biomass at concentrations from 6 uM to 24 uM.
Figure 2. Average Biomass of Sedum reflexum plants under additional copper
concentrations from 0 uM to 24 uM over time with deviations. Major deviations
occurred at Day 13 at 6 uM and Day 12 6 uM. Otherwise, weights at 6 uM and
12 uM were below 5 mg.
Day 22
*
*
*
*
**
**
**
**** ** **
**
P<0.01
*
**
**
*
+
+
+
+++
++
++ ++
*/+p<0.01
+
++
Figure 5. Left: Sedum reflexum germination at week
2 with single leaflet set development.
Right: Sedum reflexum second leaflet set at week 6.
Figure 6. Left: Ten week old Sedum reflexum visual
plant growth analysis at Day 22 copper concentration
addition. Decreased roots at 6 μM. Shortened Stem
between 6 μM and 24 μM. Whitening along the stems
and leaf curling at 24 μM
Figure 4. Average Fluorometer analysis of Sedum reflexum plants under additional
copper concentrations from 0 uM to 24 uM over time. Copper concentrations from 6
uM to 24 uM displayed a decrease overtime from Day 0 while 0 uM remained
consistent between 150 - 200 ug/g. The relative decreases between concentrations
went from 6 uM to 24 uM where 24 uM was the lowest
between 50 – 100 ug/g.
Figure 3. Average Fluorometer analysis of Sedum reflexum plants under additional
copper concentrations from 0 uM to 24 uM over time with deviations. 0 uM plants
displayed consistent chl-a levels over 22 day period. Relative chl-a concentrations
displayed a progressive decrease over time at 6 uM and 24 uM. Tukey test
indicated differences at Day 0 between Day 6,12, 22. Additionally there was
differences between Day 12 between Day 2, 5, and 13.