Supplemental text: functional categorization of differently expressed transcripts

We classified them into seven types and simplified their possible roles as following (see Table S2).

(1) Photosynthesis. All putative genes encoding components of the chloroplast and involved in photosynthesis had been down-regulated in the bracts (Table 3). For example, Rhn3592, Rhn0883, Rhn4562, and Rhn3441 are homologous to those genes related to the Photosystem II 22 kDa protein , Photosystem I reaction center subunit III, ATP synthase gamma chain 1 and Photosystem I reaction center subunit II, respectively (e.g. Haldrupet al. 2000; Liet al. 2000). In addition, Rhn3921, Rhn4885 and Rhn4282 are homologous to chlorophyll binding proteins 91R, Thioredoxin-like and NADH dehydrogenase genes, respectively (Meyer et al.1999).

(2) Stress and defense response. Many plants have developed defense mechanisms involving the production of secondary metabolites (La Camera et al. 2004). Most candidate genes related to stress and defense responsethat were identified in the present study were up-regulated in the bracts. For example, Rhn2182 is homologous to the flavonol synthase genes that take part in the synthesis of light-absorbing flavonoid compounds. These phenylalanine-derived aromatic secondary products play an important role in plants in protecting against UV-B radiation (Warrenet al. 2002; Kim et al. 2008). Another bract-specific candidate gene, Rhn0116, shows a high similarity to a myo-inositol phosphate synthase gene (MIPS protein, isomerase involved in inositol metabolism; Klig et al. 1985). Inositol is a natural cell wall osmoprotector synthesized, at the subcellular level, into phosphatidylinositol as part of a complex process and then recycled into the phosphatidyl-inositol cycle as a complex signaling mechanism under drought stress(Ishitani et al. 2002).A third mechanism concernsthe up-regulated candidate gene Rhn3552. It is homologous to glucan endo-1,3-beta-glucosidase, a peroxidase precursor that is involved in external stimuli and the control of redox balance in the cell in arid environments (Wu et al. 1994 ).

(3) Carbohydrate and secondary metabolism. Changes in metabolic activities and the expression of related genes may help specialized phenotypes to adapt to arid habitats. In bracts, Rhn5001, which is homologous to a triosephosphate isomerase (TPI), was down-regulated. This gene catalyses the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate and serves as a Calvin cycle enzyme compartmentalized in the chloroplast of higher plants (Katrin et al. 1994). The repressed TPI expressionsuggests a reduction in carbon fixation through the Calvin cycle. However, two candidate genes related to the tricarboxylic acid (TCA) cycle are up-regulated in the bracts, i.e., Rhn1194 encoding a Succinyl-CoA ligase and Rhn1031, a sucB-like gene encoding the dihydrolipoamide succinyltransferase component (Knapp et al. 2000). Succinyl-CoA ligase catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate (Kruftet al. 2001). Up-regulation of succinyl-CoA may indicate that mitochondrial activity is higher in the bracts than inthe normal leaves. In addition, Rhn0004, which is homologous to a gene to encode glutamine synthetase nodule isozyme, also increased in bracts. This enzyme plays an important role in promoting and maintaining cell function and has been found to increase under environmental stresses in other plants of arid habitats (Ndimba et al. 2005).

(4) Transcription factors and signal transduction. We found that around 12% of the differentially expressed genes are involved in signal transduction and/or gene regulation functions. For example, Rhn4886 and Rhn4883, which are homologous to the genes encoding the PHD zinc finger family proteins and 14-3-3-like proteins, respectively (Jarillo et al.1994), were down-regulated in bracts. The 14-3-3-like proteins may function as regulators in signal transduction/phosphorylation mechanisms, which were initially characterized in mammals and have been implicated in the regulation of multifunctional protein kinases (Jarillo et al.1994). However, the majority of genes in this category were up-regulated in bracts. For example, the bract-specific Rhn1632 is homologous to a MYB transcription factor (Jung et al. 2008) and this factor plays important roles in responses to environmental stimuli in Arabidopsis (Abeet al. 2003). In addition, three transcripts (Rhn4152, Rhn4805 and Rhn4273), possibly related to the calcium signaling network, are found in bracts, but not in normal leaves. Both Rhn4152 and Rhn4805 are homologous to genes that encode calmodulin-binding proteins (McCormack andBraam2003). Rhn4273 is homogenous to the CIPK9 gene encoding calcium sensor CBL-interacting protein kinase (Kolukisaoglu et al. 2004). The CIPK9 geneis a critical regulator of low potassium response in Arabidopsis plants when under stress in arid habitats (Pandey et al. 2007). Calcium signaling is known to be critically important in plant defense in arid habitats (Lecourieuxet al. 2006).

(5) Transport. Sugar and ion transporters adjust the osmotic pressure under conditions of abiotic stress (Seki et al. 2002). Our studies indicate that a few candidate genes homologous to sugar and ion transporter genes are up-regulated in bracts. For example, Rhn3593 is very similar to a sugar ERD6-like transporter.The accumulation of water soluble carbohydrates is widely regarded as an adaptive response of plants to arid conditions for maintaining leaf cell turgor, protecting membrane integrity and preventing protein denaturation (Verslues et al.2006). In addition, Rhn1032 is homologous to theNa+/H+ antiporter nhaA. This antiporter has also been found to increase in other plants in response to drought stress (e.g. Zhu et al. 2002).

(6) Protein turnover. We found that three types of candidate genes related to protein metabolism were up-regulated in the bracts. The first type involves four candidates (Rhn3554, Rhn4672, Rhn4912 and Rhn0881) that are homolognous to the genes encoding ribosomal subunits. The nuclear ribosome is an important site fortranslating nuclear proteins. The up-regulation of these ribosome genes may lead to increased synthesis of some proteins in the bracts. Under arid conditions, transcription- and translation-related proteins have been found to increase in order to synthesize defensive proteins (Ndimba et al. 2005). The second group of candidate genes involved three transcripts (Rhn3914, Rhn4352 and Rhn4913)that are homologous to those genes encoding ubiquitin proteins. These ubiquitin proteins are closely linked to substrate proteins, together playing important roles in protein degradation via the ubiquitin pathway(Jentsch and Schlenker 1995; Callis 1995). The last group of candidate genescontained two transcripts (Rhn1633 and Rhn4031) that are homologous to genes encoding heat shock proteins (HSPs). These HSPs, now known as the stress proteins, play important roles in maintaining normal physiological and cellular functions when plants are exposed to stressin arid habitats (Grover 2002). In contrast, a few transcripts related to protease were repressed in the bracts, namely Rhn2281 and Rhn4722, which are homologous, respectively, to genes encoding ATP-dependent Clp protease and proline iminopeptidase. In addition, a pair of transcripts, Rhn3561 and Rhn3554), probably also encoding a similar ribosomal protein were differentially expressed in the bracts and normal leaves respectively.

(7) Cytoskeleton and cell wall biosynthesis. The bract-specific transcripts include those homologous to the histone genes (Rhn4631 and Rhn4193), to the tubulin gene (Rhn4432), to the pectate lyase gene (Rhn4281) and to the glucose-6-phosphate 1-dehydrogenase (G6PD) gene (Rhn3913). Histone is the core component of nucleosome and plays a central role in transcription regulation, DNA repair and replication as well as chromosomal stability (Okada et al. 2005). Meanwhile, tubulin proteins are involved in cytoskeleton development, as a result of which cell expansion and cell plates are accurately positioned during plant morphogenesis (Kost and Chua 2002). Pectate lyases, also known as pectate transeliminases, catalyse the eliminative cleavage of de-esterified pectin, which is a major component of the primary cell walls of plants (Sakamotoet al. 1994).G6PD is referred to as the first and rate limiting enzyme of the pentose phosphate pathway,providing reducing power to all cells in the form of NADPH, thus playing important roles in the biosynthesis of the sugar moiety of nucleic acids during cell wall synthesis (Reiter 2008). In addition, this protein hasbeen found to take part in plant defense against oxidative stress (Pandolfil et al. 1995). However, Rhn4002, which is homologous to a histone-lysine N-methyltransferase ATXR5 gene, appeared in normal leaves, but was absent from the bracts. This gene plays an important role in the cell cycle or DNA replication (Ce´ cile et al. 2006).

References

Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15:63–78

Callis J (1995)Regulation of protein degradation.Plant Cell 7:845-857

Ce´cile R, Rosangela S,, Nathalie G, Se´ verine D, Claudette P, Rino C, Eva K, Catherine B (2006)Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis Plant. J 47: 395–407

Grover A (2002) Molecular biology of stress responses. Cell Stress Chaperon1:1-5

Haldrup A, Simpson DJ, Scheller HV (2000) Down-regulation of the PSI-F subunit of photosystem I (PSI) in Arabidopsis thaliana The PSI-F subunit is essential for photoautotrophic growth and contributes to antenna function. J BiolChem275:31211–31218

Ishitani M, Majumder AL, Bornhouser A, Michalowski CB, Jensen RG, BohnertHJ (2002) Coordinate transcriptional induction of myo-inositol metabolism during environmental stress. Plant J9:537–548

Jarillo JA, Capel J, Leyva A, Martinez Zapater JM, Salinas J (1994) Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators.Plant MolBiol 25:693–704

Jentsch S, Schlenker S (1995) Selective protein degradation: a journey's end within the proteasome.Cell 82:881–884

Jung C, Seo JS, Han SW, Koo YJ, Kim CH, Song SI, Nahm BH, Choi YD, Cheong JJ (2008) Overexpression of AtMYB44 enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis.Plant Physiol 146:623–635

Katrin H, Claus S, Josef K, William M (1994)Chloroplast and cytosolic triosephosphate isomerases from spinachpurification, microsequencing and cDNA cloning of the chloroplast enzyme. Plant Mol Biol26: 1961-1973

Kim BG, Kim JH, Kim J, Lee C, Ahn JH (2008) Accumulation of flavonols in response to ultraviolet-B irradiation in soybean is related to induction of flavanone 3-beta-hydroxylase and flavonol synthase.Mol Cells 25:247–52

Klig LS, Homann MJ, Carman GM,Henry SA (1985) Coordinate regulation of phospholipid biosynthesis in Saccharomyces cerevisiae: pleiotropically constitutive opi1 mutant.J Bacteriol 162:1135–1141

Knapp JE, Carroll D, Lawson JE, Ernst SR, Reed LJ, Hackert ML (2000) Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase.Protein Sci 9:37-48

Kolukisaoglu U, Weinl S, Blazevic D, Batistic O, Kudla J(2004) Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL-CIPK signaling networks .Plant Physiol 134:43–58

Kost B, ChuaNH (2002) The plant cytoskeleton: vacuoles and cell walls make the difference. Cell 108:9–12

Kruft V, Eubel H, Jaensch L, Werhahn W, Braun HP (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis.Plant Physiol127:1694–1710

La Camera S, Gouzerh G, Dhondt S, Hoffmann L, Fritig B, Legrand M , Heitz T (2004) Metabolic reprogramming in plant innate immunity: the contributions of phenylpropanoid and oxylipin pathways.Immunol Rev198:267–284

Lecourieux D, Ranjeva R, Pugin A (2006) Calcium in plant defence-signalling pathways. New Phytol 171:249–269

Li XP, Bjorkman O, Shih C, Grossman AR, Rosenquist M, Jansson S, Niyogi KK(2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting.Nature 403:391–395

McCormack E, Braam J (2003) Calmodulins and related potential calcium sensors of Arabidopsis.New Phytol 159:585–598

Meyer Y, Verdoucq L, Vignols F (1999) Plant thioredoxins and glutaredoxins: identity and putative roles.Trends Plant Sci 4:388–394

Ndimba BK, Chivasa S, Simon WJ, SlabasAR (2005) Identification of Arabidopsis salt and osmotic stress responsive proteins using two-dimensional difference gel electrophoresis and mass spectrometry. Proteomics 5:4185–4196

Okada T, Endo M, Singh MB, Bhalla PL(2005)Analysis of the histone H3 gene family in Arabidopsis and identification of the male-gamete-specific variant AtMGH3. Plant J 44:557–568

Pandey GK, Cheong YH, Kim BG, Grant JJ, Li L, Luan S(2007) CIPK9: a calcium sensor-interacting protein kinase required for low-potassium tolerance in Arabidopsis. Cell Res 17:411–421

Pandolfil PP, Sonati F, Rivil R, Mason P, Grosveld F, Luzzattol L (1995) Targeted disruption of the housekeeping gene encoding glucose 6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO J 14:5209–5215

Reiter WD (2008) Biochemical genetics of nucleotide sugar interconversion reactions.Curr Opin Plant Biol 11:236–243

Sakamoto T, Hours RA, Sakai T (1994) Purification, characterization, and production of two pectic transeliminases with protopectinase activity from Bacillus subtilis. BiosciBiotech Bioch 58:353–358

Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, Satou M, Akiyama K, Taji T, Yamaguchi-Shinozaki K, Carninci P, Kawai J, Hayashizaki Y, Shinozaki K (2002) Monitoring the expression profiles of 7000 Arabidopsisgenes under drought, cold and high-salinity stresses using afull-length cDNA microarray.Plant J31:279–292

Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK (2006) Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J45:523–539

Warren JM, Bassman JH, Mattinson DS, Fellman JK, Edwards GE, Robberecht R (2002) Alteration of foliar flavonoid chemistry induced by enhanced UV-B radiation in field-grown Pinus ponderosa, Quercus rubra and Pseudotsuga menziesii.J Photoch Photobio B66:125–133

Wu S, Kriz AL, Widholm JM(1994) Nucleotide sequence of a maize cDNA for a class II, acidic beta-1,3-glucanase. Plant Physiol106:1709–1710

Zhu J (2002) Salt and drought stress signal transduction in plants.Annu Rev Plant Biol53:247–273

1