1. Large Expression Differences in Genes for Iron and Zinc Homeostasis, Stress Response, and Lignin Biosynthesis Distinguish Roots of Arabidopsis thaliana and the Related Metal Hyperaccumulator Thlaspi caerulescens Judith E. van de Mortel, Laia Almar Villanueva, Henk Schat, Jeroen Kwekkeboom, Sean Coughlan, Perry D. Moerland, Emiel Ver Loren van Themaat, Maarten Koornneef, and Mark G.M. Aarts Plant Physiology (2006) November; 142(1) 1127–1147 BioInformatics Lab Tuesday, April 13, 2010 Kristoffer Chin Salomon Garcia Michael Piña

2. Outline Introduction – Micronutrient Zinc – Arabidopsis thaliana and Thlaspi caerulescens – Lignin Biosynthesis Materials and Methods – Microarray Experiment – RT-PCR Results – Between A. thaliana and T. caerulescens, 2,200+ genes were significantly differentially expressed Discussion – High expression of lignin biosynthesis genes corresponds to lignin deposits in the endodermis Future studies References

3. Micronutrients are essential for humans, plants, and animals Zinc plays an important role in plants’ biological processes (co-factor for 300+ enzymes) Toxic in high amounts – Plants keep tight regulation over zinc homeostasis Zinc homeostasis mechanism is generally universal within plants with a few exceptions

4. Zinc hyperaccumulators can accumulate large amounts of zinc More than 1% of their dry weight 10,000 µg zinc g⁻¹ in hyperaccumulators 30-100 µg zinc g⁻¹ in most other plants – Over 300 is usually toxic T. caeulescens is a zinc hyperaccumulator – Very closely related to A. thaliana Zinc conc. in shoots is often higher than in roots in hyperaccumulators

5. A. thaliana and T. caerulescens share 88.5% DNA identity in coding regions A. thalianaT. caerulescens schaechter.asmblog.org

6. A complex network of homeostatic mechanisms has evolved in plants These mechanisms control the uptake, accumulation, trafficking, and detoxification of metals – This also applies for hyperaccumulators The physiology of metal hyperaccumulation is well understood, but the molecular genetics have not been explored in detail

7. Which genes are most likely relevant for adaptation to high zinc exposure in T. caerulescens? Examine the response of roots to zinc deficiency, sufficiency, and excess DNA microarray covering nearly the entire transcriptome of A. thaliana – All available annotated genes – ~10,000 non-annotated genes Comparison of A. thaliana to T. caerulescens

8. Plants were prepared for a root and shoot metal accumulation assay Arabidopsis thaliana Columbia-0 Thlaspi caerulescens J. & C. Presl accession La Calamine Germinated on garden peat soil 3 week old seedlings transferred to pots containing half strength Hoagland solution pH buffer was added and the pH was set at 5.5 After 3 weeks, both species were transferred to the modified Hoagland solution containing: – Deficient (0 µM) ZnSO₄ – Sufficient (100 µM) ZnSO₄ – Excess (1,000 µM) ZnSO₄

9. After 4 weeks of growth, plants were harvested Root system was desorbed with cold 5mM PbNO₃ Roots and shoots were dried overnight Wet-ashed – Mixture of HNO₃ and HCL Analyzed for zinc, iron, and manganese using flame atomic absorption spectrometry

10. cDNA from T. caerulescens roots exposed to sufficient (100 µM) zinc was used as the common reference for the microarray The common reference was labeled with Cy3, treatment samples were labeled with Cy5 – Dye-swap used for quality control (QC) Roots of one pot were pooled and homogenized in liquid nitrogen Each pool (3 plants of either species) was considered as 1 biological replicate – 2 biological replicates were used

11. RNA was extracted, purified, and hybridized on slides for the microarray 27,000+ annotated genes & 10,000+ nonannotated genes After hybridization, slides were scanned, analyzed, normalized – Agilent Feature Extraction software Moderated t test to find differentially expressed genes – P values were considered to be significant if they were <0.05 Significantly differentiated genes were clustered in a tree diagram Dye-swap hybridization was performed for QC

12. RNA for both species was extracted for semiquantitative RT-PCR New primers were created to ensure the correct amplification for T. caerulescens genes – MMLV reverse transcriptase Care was taken in creating primers for Arabidopsis to ensure comparable positions and lengths as T. caerulescens 25-35 PCR cycles

13. Three conditions were designed to expose the zinc in A. thaliana Sufficient condition: 2 µM ZnSO4 – no phenotypic differences with deficient condition Deficient condition: 0 µM ZnSO4 – no phenotypic differences with sufficient condition Excess condition: 25 µM ZnSO4 – Little growth inhibition in the roots

14. The three conditions for T. caerulescens were different due to hyperaccumulation Sufficient condition:100 µM ZnSO4 Deficient condition: 0 µM ZnSO4 Excess condition:1 mM ZnSO4 – No altered phenotype in all conditions

15. Differences in mineral concentration of zinc, iron, and manganese in roots and leaves

16. Genes of A. thaliana from microanalysis was organized into four clusters; 608 genes Identified Cluster ICluster IICluster IIICluster IV # of genes found 98 genes128 genes347 genes35 genes Condition found in Sufficient and excess ExcessDeficientDeficient and Sufficient Functions stress response Metabolism Heat schock proteins 15 with unkown function 20 not annotated Iron homeostasis Metal transporters Stress response Metabolism Transcription factors Metal homeostasis Metal transporter Protein stability Signal transduction Transcription regulation Metabolism 164 genes unknown Secondary metabolism Biotic stress response Transcription 5 genes with unkown function

17. 220 genes did not hybridize with T. caerulescens DNA 85% - 90% DNA similarity in A. thaliana and T. caerulescens Verified hybridization first with the use of sufficient conditions on both plants Spot intensities were better in A. thaliana than T. caerulescens The 220 genes were excluded from data

18. Genes of T. caerulescens from microanalysis was organized into six clusters; 350 genes Identified Cluster ICluster II Cluster IIIA Cluster IIIB Cluster IVA Cluster IVB Others # of genes found 38 with II38 with I74161914189 Condition found in Deficient Excess Sufficient Functions ZIP genes Metal homeostasis Lignin biosynthsis ZIP genes Metal homeost asis Lignin biosynth sis Oxidative stress response Senescens e Ehylene biosynthesi s Plant defense Oxidative stress response Senescen se Ehylene biosynthes is Plant defense Little metal homeostas is General metabolis m Stress response Mostly unkown

19. 2,272 genes were found to be highly express in T. caerulescens than A. thaliana A. thaliana 420 genes not expressed in root Little variation in expression among conditions Less expression in PDF genes Less lignin biosynthesis genes Less cellular process Less transport process Less stress response Less transcription T. caerulescens 420 genes expressed in root High variation variation in expression among conditions More expression in PDF genes More lignin biosynthesis genes More cellular process More transport process More stress response More transcription

20. The larger amount of genes in lignin biosynthesis should be physically seen between the plants

21. Semiquantititaive Reverse Transcription-PCR used to confirm the genes expressions found in the microarray between the two plants

22. Zinc homeostasis found differential along with iron accumulation T. caerulescens is able to maintain nontoxic zinc levels while translocating high amounts of zinc to the leaves An unexpected event occurs and that is that iron accumulates in the roots of Arabidopsis and T. caerulescens at increasing zinc concentrations The effect found in both species suggests that the increase in iron uptake is due to prevent possible risks of iron deficiency in leaves.

23. Genes involved in zinc homeostasis are highly expressed in deficiency than other conditions Some genes known to be involved in zinc homeostasis are ZIP2, 4, 5 and 9, NAS2 and HMA2 genes Highly expressed in zinc deficiency include ZIP1, 3, and 10, IRT3, MTP2, and NAS4 These transporters are involved in the transport of cations across plasma membrane. Not all of them are involved in the uptake of zinc in the same tissue. It is likely that these transporters do similar functions in different parts of the roots or are found in intracellular membrane.

24. NAS and YSL genes and their ability to be induced by zinc deficiency NAS2 and NAS4 genes are highly expressed in roots under deficiency rather than sufficiency. YSL genes are also induced by zinc deficiency. YSL genes are implicated in the transport of NA metal chelates within the plant and the entry of metals to the phloem and xylem. YSL2 and YSL3 are slightly affected by different zinc treatments, which in turn lead them to find that genes were slightly induced by lower zinc concentrations.

25. High zinc deficiency-induced expression of FRD3, FRO4, and FRO5 Arabidopsis was grown under zinc deficient conditions This was a significant observation because FRD3 has been known to be implicated for the most part with iron homeostasis. FRO4 and FRO5 approximate the ferric chelate reductase gene FRO2, in the contrary to FRO2 their expression was not induced in the root of Arabidopsis upon iron deficiency.

26. 128 genes of Arabidopsis was more highly expressed in excess The expression of these genes is related to a defense against oxidative stress caused by the treatment of zinc. Large fraction of these genes was found to have an expressed comparison between wild type Arabidopsis and fit1 mutant.

27. T. caerulescens has a smaller differential in genes For this species the response to zinc deficiency and zinc excess is quite different from Arabidopsis. Unlike Arabidopsis, T. caerulescens expresses the ZIP family (ZIP3, 4, and 9) under zinc sufficient conditions Similar to Arabidopsis, T. caerulescens also expresses a cluster of genes in zinc deficient conditions, but this cluster is quite smaller. The probable cause for this is differences in hybridization efficiency.

28. NAS genes and their importance to T. caerulescens The expression of these three NAS genes in T. caerulescens suggest that they are a major function for metal adaptation. The presence of these genes indicates that there will be flexibility when it comes to NAS gene expression

29. Genes expressed differently from T. caerulescens and Arabidopsis There are more than 2200 genes which are quite significant and differentially expressed in the three zinc treatments 50% of the genes found in T. caerulescens are of unknown function. Stress respond genes expressed in T. caerulescens are different from Arabidopsis.

30. Expressed genes that were found and biological role is unclear Several expressed genes were 100-fold on T. caerulescens. These genes were defensin genes or PDF genes These genes included 15 genes which 4 were PDF genes. One of these PDF genes included one that was close to being 1000-fold which was expressed in both deficient and excess zinc. (PDF1.1) The biological role of defensin is unclear.

31. Arabidopsis had low expression of PDF genes Jasmonic acid (stress hormone) is responsible for the expression of PDF1.2 in Arabidopsis. Heavy metal stress is involved in the accumulation of jasmonic acid. – PDF1.2a, 1.2b, 1,2c, and 1.3 are induced upon potassium starvation. These genes suggest a relation between jasmonic signaling and potassium starvation. 46 genes were found to be more highly expressed in T. caerulescens than in Arabidopsis – Potassium transporter genes HAK5, KUP3, and KAT, were found to be highly expressed in T. caerulescens.

32. Zinc hyperaccumulation trait in T. caerulescens and the required expression of metal hyperaccumulation genes Considered 16 highly expressed genes at 100 micrometer ZnSO 4, of which 4 were known to be Zinc transporters HMA4, MTP1, ZIP1, and IRT3 – In the study conducted by Papoyan and Kochian HMA4 was identified to being involved in zinc hyperaccumulation, particular loading of zinc into the xylem. Other Zinc transporters that were highly expressed in T. caerulescens included HMA3, MTP8, and NRAMP3. – HMA3 is similar to HMA4, and in Arabidopsis the expression of this gene is not affected by exposure to zinc

33. Genes found to contribute in the movement of zinc The MTP8 gene is a member of the cation diffusion facilitator family. – This gene was found to be highly expressed in T. caerulescens at sufficient and deficient conditions compared to Arabidopsis. AtNRAMP2 is a vacuolar transporter is able to transport cadmium and iron. The induction of TcNRAMP3 gene expression by deficiency of zinc suggests it has an important role in the movement of zinc and iron in T. caerulescens and Arabidopsis.

34. Iron homeostasis genes and the unexpected outcome that resulted between T. caerulescens and Arabidopsis IRT1, IRT2, and FRO2 (iron homeostasis genes) were not induced in T. caerulescens upon excess treatment of zinc. Testing with RT-PCR did not detect the expression of TCIRT1 except in roots at lower levels of zinc. This suggests that T. caerulescens is able to regulate its iron and zinc homeostasis independently, unlike Arabidopsis, or that continued expression of zinc transporters enables low efficiency but enough iron uptake in T. caerulescens.

35. Expression of similar genes and the different outcome between T. caerulescens and Arabidopsis High expression of 24 genes suggested a function in lignin biosynthesis, and 13 genes are involved in suberin biosynthesis in T. caerulescens. These genes included (CER3, CER6,and 11 LTP genes) CER3 is known to be expressed in the roots of Arabidopsis, but the expression of similar gene CER6 in the roots of T.caerulescens is quite different. High expression of lignin and suberin biosynthesis concurs well with the U-shaped lignification and suberinization of the endodermis cells and the occasional presence of second endodermal layer found in the roots of T.caerulescens. This U-shape apparenece is uncommon in plants, this usually occurs at older sections of the root hairs when they are no longer active. Suggesting that this layer helps to prevent the eflux of metals from the vascular cylinder.

36. Genes differentially expressed certain alterations in transcript levels may occur 131 transcriptional regulators with more than 5-fold higher expression were found in T. caerulescens Of the 19 genes that were more than 10-fold higher expressed in zinc deficient conditions 2 genes (INO and SPL) are said to be involved with the development of flowers in Arabidopsis but expression is irregular. Similar to this irregular expression it was also found that FIS2 gene is more highly expressed in the roots of T.caerulescens. – In Arabidopsis, this gene is expressed in the development of seeds. – This gene is found in A. halleri and is induced in the response to the exposure of zinc.

37. Comparative analyses show T. caerulescens and Arabidopsis show role in zinc homeostasis genes and there adaptation to high zinc exposure The analysis also suggests that there are many uncharacterized genes with similar functions and that there are many that are unaccounted for and their functions are unknown. While some of the genes were differentially expressed between A. halleri and Arabidopsis: – Many of them were not at different levels – Thus, suggesting an overlap in mechanisms of metal accumulation and tolerance.

38. References van de Mortel JE, Almar Villanueva L, Schat H, Kwekkeboom J, Coughlan S, Moerland PD, Ver Loren van Themaat E, Koornneef M, and Aarts MG. Large expression differences in genes for iron and zinc homeostasis, stress response, and lignin biosynthesis distinguish roots of Arabidopsis thaliana and the related metal hyperaccumulator Thlaspi caerulescens. Plant Physiol 2006 Nov; 142(3) 1127-47.