1. Characterization of gig1 (glucose insensitive growth 1) Reveals the Involvement of the Plastidic Copper Transporter PAA1 in Sugar-mediated Interorganellar Communication Shin Ae Lee, Jun Lim Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Korea It is known that high glucose (6%) conditions inhibit growth of the Arabidopsis seedlings. Based on the growth-arrested phenotype, we have screened ~1500 activation-tagged seedlings for insensitive growth in the presence of 6% glucose. We identified a recessive mutant with strong insensitive growth, named gig1 (glucose insensitive growth 1). Under standard growth conditions, gig1 mutants exhibited a short-root phenotype. Thus, we characterized developmental defects of gig1 with tissue-specific markers for the specification and maintenance of stem cell niche. Although no patterning defects were found in gig1, root meristem size was drastically reduced and cell division potential monitored by CYCB1;1-glucuronidase (GUS) reporter was also severely reduced. To understand the molecular basis of gig1 in glucose signaling, we isolated the GIG1 gene by TAIL-PCR, and found a T-DNA insertion in the PAA1 locus, which is previously known as a P-Type ATPase that transports copper (Cu) to the chloroplast stroma. We also confirmed that growth of gig1 was complemented with Cu supplement in MS agar plates, and the genomic clone including the promoter region and ORF rescued gig1 phenotypes. In addition, another recessive T-DNA insertion allele from the SIGNAL database exhibited the identical phenotype. To elucidate the role of GIG1 in sugar signaling, we performed genetic analysis with hexokinase 1 (hxk1) and aba-insensitive 4 (abi4). Our findings indicate that GIG1 is epistatic to both HXK1 and ABI4 in glucose signaling, suggesting that there is sugar-mediated interorganellar communication. (glucose) 1% 6%1%6% WT gig1 1% 6%1%6% WT gig1 A B C D E WT gig1 WTgig1 (DAG) WT gig1 (DAG) WT gig1 WT gig1 WTgig1 WTgig1 F G 4x35Se SALK_043208 1kb barLB RB A B gig1-1 gig1-2 WTgig1-1gig1-2 ABCD E F - gig1 (glucose insensitive growth 1) specifically exhibits insensitive phenotype under high glucose conditions. - GIG1 encodes the plastidic copper transporter PAA1. - GIG1 is epistatic to hxk1 and abi4. - Reduction of GIG1 expression in hxk1 and abi4 indicates that its expression is subject to regulation by nuclear genes HXK1 and ABI4. - GIG1 plays a role in interorganellar crosstalk between nucleus and plastid. - Our findings indicate that a bidirectional signaling exists in the presence of high glucose. Figure 1. Root growth of gig1 under high sugar conditions. (A) gig1 shows insensitive phenotype under 6% glucose conditions. Root length of gig1 and WT (B), and anthocyanin contents (C) under 6% glucose. Growth of gig1 and WT under 12% sucrose(D), and 300mM mannitol (E). WT gig1 WTWT WT gig1 WT gig1 WT gig1 AB C ED E Figure 2. Expression study of sugar signaling markers. ABI3, ABI4, ABI5, ApL3 and CHS genes are upregulated by high glucose in WT, whereas induction of marker genes is drastically reduced in gig1. Figure 3. gig1 phenotype under normal conditions. (A),(B) Root length of gig1 is shorter than that of WT on 1% glucose media. (C)-(E) Meristem size and cortex cell number of gig1 are reduced compared to those in WT. Bottom arrowheads show QC (quiescent center) and upper arrowheads indicate the cortex transition zone. (F),(G) CYCB1;1::GUS expression showing mitotic cell division is reduced in gig1. Summary Figure 4. Identification of the GIG1 gene. (A) GIG1 encodes the plastidic copper transporter PAA1. Two T-DNA insertion alleles are depicted. (B) Two gig1 mutations are likely null, shown by RT-PCR. (C) Domain of PAA1 protein (from Abdel- Ghany et al., 2005). Figure 5. Expression study of GIG1. pGIG1::GUS is expressed broadly at cotyledons and leaves and root vasculature. (A) Whole seedling. (B) Differentiation zone of the root. (C) Cross section of the root. (D) Root tip. (E) Expression levels of GIG1 in different tissues. (F) Expression level of GIG1 in 1% and 6% glucose conditions. Figure 6. Supplement of copper rescues the gig1 mutant phenotype in normal conditions. (A),(B) Root length of 12 DAG seedlings. (C),(E),(F) Meristem cell number and length. (D),(G) CYCB1;1::GUS expression. WTgig1 WT gig1 WT gig1 WT gig1 WTgig1 WTgig1WTgig1 A BCD E F G 1% 6%1%6% WT gig1 WT gig1 +CUSO 4 1% 6%1%6% WT gig1 +CUSO 4 A B C Figure 7. Supplement of copper cannot rescue the gig1 mutant phenotype in 6% glucose conditions. (A),(B) Root length of 12 DAG seedlings. (C) Induction of sugar signaling markers are not properly restored by copper. gig1 abi4 gig1xabi4gig1xhxk1hxk1WT gig1 abi4 gig1xabi4gig1xhxk1hxk1WT Nucleus Plastid Anterograde signaling Retrograde signaling GUN1 ABI4 GIG1/ PAA1 Cu LHCB RBCS Glucose ROS HXK1 Cu A B CD Figure 8. gig1 is epistatic to hxk1 and abi4. (A) 12 DAG seedlings in the presence of 1% glucose. (B) 12 DAG seedlings in the presence of 6% glucose. (C) GIG1 expression is up regulated by glucose, whereas induction of GIG1 is drastically reduced in hxk1 and abi4. (D) Scheme of interorganellar communication between nucleus and chloroplast.