N代谢的重要调节因子----中国科学院西双版纳热带植物园.pdf
N代谢的重要调节因子----中国科学院西双版纳热带植物园.pdf
The SNAC-A Transcription Factor ANAC032 Reprograms Metabolism in Arabidopsis Liangliang Sun1,4, Ping Zhang1,2,4, Ruling Wang1,4, Jinpeng Wan1,2, Qiong Ju1, Steven J. Rothstein3 and Jin Xu1,* CAS Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun, Mengla, Yunnan 666303, China 2 University of Chinese Academy of Sciences, Beijing 100049, China 3 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada 4 These authors contributed equally to this work. Studies have indicated that the carbon starvation response leads to the reprogramming of the transcriptome and metabolome, and many genes, including several important regulators, such as the group S1 basic leucine zipper transcription factors (TFs) bZIP1, bZIP11 and bZIP53, the SNAC-A TF ATAF1, etc., are involved in these physiological processes. Here, we show that the SNAC-A TF ANAC032 also plays important roles in this process. The overexpression of ANAC032 inhibits photosynthesis and induces reactive oxygen species accumulation in chloroplasts, thereby reducing sugar accumulation and resulting in carbon starvation. ANAC032 reprograms carbon and nitrogen metabolism by increasing sugar and amino acid catabolism in plants. The ChIP-qPCR and transient dual-luciferase reporter assays indicated that ANAC032 regulates trehalose metabolism via the direct regulation of TRE1 expression. Taken together, these results show that ANAC032 is an important regulator of the carbon/energy status that represses photosynthesis to induce carbon starvation. Keywords: ANAC032 Metabolism Photosynthetic inhibition Stress response TRE1 Trehalose. Introduction The Arabidopsis ANAC032 gene encodes the SNAC-A (subfamily) member of the stress-responsive NAC [No Apical Meristem (NAM), Arabidopsis ATAF1/2 and Cup-shaped Cotyledon2 (CUC2)] transcription factors (TFs). The phytohormones abscisic acid (ABA), salicylic acid (SA) and methyl jasmonate (MeJA) induce ANAC032 expression (Nakashima et al. 2012, Allu et al. 2016, Mahmood et al. 2016a, Mahmood et al. 2016b). Ectopic overexpression of ANAC032 in Arabidopsis induces age-dependent and stress-induced leaf senescence, alters responses to auxin and results in hydrogen peroxide (H2O2) accumulation in plants (Mahmood et al. 2016a). Salt and osmotic stresses markedly induce the expression of ANAC032, and overexpression of ANAC032 increases sensitivity to salt and drought. In contrast, the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst) also induces ANAC032 expression, but overexpression of ANAC032 increases resistance to Pst, suggesting that opposite roles of ANAC032 in modulating biotic and abiotic stress responses exist (Nakashima et al. 2012, Allu et al. 2016, Mahmood et al. 2016a, Mahmood et al. 2016b). ANAC032 improves pathogen-induced defense responses by activating SA signaling and represses Coronatine (COR)-mediated stomatal reopening by repressing jasmonic acid (JA) signaling. ANAC032 regulates both SA and JA signaling by regulating the expression of MYC2, NIMIN1 and PDF1.2A by binding to their promoters (Allu et al. 2016). Previous studies have shown the functional redundancy of SNAC-A TFs in modulating ABA-induced leaf senescence (Takasaki et al. 2015). Mutants with a single mutation in the ANAC032 gene did not present a distinct phenotype during age-dependent and osmotic stress-induced leaf senescence (Vermeirssen et al. 2014, Takasaki et al. 2015, Mahmood et al. 2016a). However, chimeric repressor (SRDX32) lines are significantly tolerant to salt and drought stress and present reduced reactive oxygen species (ROS) accumulation and oxidative damage in leaves (Mahmood et al. 2016a). In these repressor lines, the C-terminal domain of ANAC032 is fused to a plantspecific ERF-associated amphiphilic repression (EAR); modified EAR motif plant-specific repression domain showing strong repression activity (SRDX); the phenotype of these lines is similar to that of loss-of-function mutants, as the fusion protein suppress the expression of target genes by overcoming the functionally redundant TFs (OshiMa et al. 2011, Mahmood et al. 2016a). The control of metabolism is essential for plant growth and development, especially during stress responses. The sucrose nonfermenting-1 related protein kinase 1 (SnRK1)-bZIP11-trehalose 6-phosphate (T6P) regulatory circuit plays a crucial role in modulating carbon starvation responses by perceiving and integrating metabolic signals to regulate plant growth (Paul 2008, Smeekens et al. 2010). Garapati et al. (2015a) reported that ATAF1, which is homologous to ANAC032, directly regulates TREHALASE1 (TRE1) expression and triggers sucrose starvation-type changes within the global transcriptome and during primary metabolism. Both ATAF1 and ANAC032 are markedly induced by carbon starvation (Bläsing et al. 2005); however, Plant Cell Physiol. 60(5): 999–1010 (2019) doi:10.1093/pcp/pcz015, Advance Access publication on 25 January 2019, available online at https://academic.oup.com/pcp ! The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 *Corresponding author: E-mail, xujin@xtbg.ac.cn; Fax, 0086-871-65140420. (Received October 30, 2018; Accepted January 11, 2019) Regular Paper 1 L. Sun et al. | ANAC032 is a regulator of carbon/energy status whether ANAC032 is also involved in modulating carbon starvation responses remains unknown. Here, we show that ANAC032 plays important roles in regulating metabolism in plants. The transcriptome and physiological analyses revealed that ANAC032 represses photosynthesis and induces ROS accumulation, thereby reducing sugar accumulation in plants. ANAC032 also induces carbon starvation responses by directly activating TRE1 expression to trigger sugar and amino acid catabolism, thereby maintaining the energy supply and survival in plants under environmental stresses. ANAC032 inhibits photosynthesis and induces ROS accumulation in leaves Previous studies have shown that ANAC032 overexpression OX32 plants accumulate more biomass, whereas the chlorophyll content in chimeric repressor of ANAC032 line SRDX32 plants is higher than that in wild-type plants (Mahmood et al. 2016a, Mahmood et al. 2016b; Fig. 1A). We therefore were interested in whether ANAC032 affects photosynthesis. To address this question, we chose the overexpression lines OX32.6 and OX32.8 and the chimeric repressor of ANAC032 lines SRDX32.1 and SRDX32.6 in this study (Mahmood et al. 2016a). First, we measured the photosystem II (PSII) activity Fig. 1 Overexpression of ANAC032 inhibits photosynthesis. (A) Leaf phenotype and chlorophyll contents of 6-week-old soil-grown Col-0, OX32 and SRDX32 plants. (B) Fv/Fm, PSII, PQ pool redox status and NPQ were measured in soil-grown Col-0, OX32 and SRDX32 plants. n = 8. The error bars represent the SE, and the asterisks indicate significant differences from the control (Student’s t-test, P < 0.01). (C) ANAC032 activation represses the expression of key photosynthetic genes. Heat maps indicate log2-fold change in the expression of genes involved in photosystem assembly relative to Col-0. (D) ANAC032 induces ROS accumulation in chloroplasts. Six-week-old soil-grown Col-0, OX32 and SRDX32 plants were maintained under light for 8 h. O2 and H2O2 were visualized by nitro blue tetrazolium (NBT) and 3,30 -diaminobenzidine (DAB) staining, respectively. 1000 Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 Results using chlorophyll fluorescence in intact leaves (Fig. 1B). The maximum quantum yield Fv/Fm and the effective quantum yield PSII are significantly lower in the OX32 lines but higher in the SRDX32 lines. We also monitored 1-qL, which reflects the plastoquinone (PQ) redox state of PSII, and found that 1-qL is higher in the SRDX32 lines and lower in the OX32 lines, implying a functional impairment of PSII in OX32 leaves. To further elucidate the molecular mechanisms underlying the ANAC032-mediated inhibition of photosynthesis, we performed the RNA sequencing (RNA-seq) to analyze the differential expressed genes (DEG). Because OX32.6 and OX32.8 lines showed high similar phenotype (Fig. 1 and Mahmood et al. 2016a), the SRDX32.6 showed similar phenotypes while stronger than those in SRDX32.1 (Fig. 1 and Mahmood et al. 2016a), we thus chose OX32.8, SRDX32.1 and SRDX32.6 lines in the transcriptome analysis. We analyzed the RNA-seq data from 8-day-old Arabidopsis Col-0, OX32.8, SRDX32.1 and SRDX32.6 seedlings with three biological replicates of each line (Supplementary Materials and Methods). The DEG were identified by comparing to the wild-type Col-0 control plants (log2-fold change 2 and adjusted P-value (Q-value) 0.001), and we obtained 1,844, 305 and 2,723 DEGs in the OX32.8, SRDX32.1 and SRDX32.6 plants, respectively (Supplementary Tables S1–S3). We then performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification (Supplementary Fig. S1) and functional enrichment (Supplementary Fig. S2) Plant Cell Physiol. 60(5): 999–1010 (2019) doi:10.1093/pcp/pcz015 ANAC032 triggers the carbon starvation response and induces the expression of genes involved in autophagy and endoplasmic reticulum (ER) stress responses A previous study has shown that both ANAC032 and ATAF1 are markedly induced by carbon starvation (Bläsing et al. 2005), and ATAF1 triggers carbon starvation responses in plants (Garapati et al. 2015a). The above results indicated that ANAC032 represses photosynthesis and reduces sugar accumulation in plants; therefore, we speculated whether ANAC032 also triggers carbon starvation responses in plants. To address this question, we investigated the possible involvement of ANAC032 in carbon starvation responses using the methods described by Garapati et al. (2015a). We investigated carbon fixation-regulated genes in the OX32.8, SRDX32.1 and SRDX32.6 lines and found that the genes repressed by carbon fixation were induced in the OX32.8 plants and vice versa (Fig. 3A, B; Supplementary Fig. S4B and Supplementary Table S4) (Bläsing et al. 2005, Garapati et al. 2015a). This transcriptome data analysis implied that similar to ATAF1, ANAC032 induces carbon starvation responses. Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 based on the DEG results. The DEG were enriched in photosynthesis, starch and sucrose metabolism and phytohormone signal transduction. In particular, the transcriptome data indicated that the expression of the genes encoding the subunits of photosystem I (PSI) and PSII proteins were largely downregulated in the OX32.8 plants, whereas these genes were upregulated in the SRDX32.1 and SRDX32.6 lines, especially in SRDX32.6 plants (Fig. 1C). Inhibition of photosynthesis could lead to ROS accumulation (Su et al. 2018). We therefore examined ROS accumulation in the leaves. Consistent with the results of Mahmood et al. (2016), the accumulation of O2 and H2O2 in the chloroplasts of the OX32 lines were higher than that in the Col-0 plants (Fig. 1D). Taken together, the results show that ANAC032 inhibits photosynthesis and thereby induces ROS accumulation in leaves. The inhibition of photosynthesis disrupted sugar accumulation in plants (Apel and Hirt 2004). The KEGG pathway functional enrichment results also indicated that ANAC032 affects sugar metabolism (Supplementary Fig. S2). Therefore, we examined the levels of soluble sugars (glucose, fructose and sucrose) and starch in the OX32 and SRDX32 lines (Fig. 2A, B and Table 1). The overexpression of ANAC032 slightly but significantly reduced the contents of glucose and sucrose, whereas the SRDX32 plants exhibited significantly increased contents of glucose, fructose and sucrose (Fig. 2A and Table 1). Overexpression of ANAC032 reduced starch accumulation, whereas the SRDX32 plants presented increased starch accumulation (Fig. 2B and Supplementary Fig. S3). Consistent with the reduced starch content in the OX32 plants, the expression of the starch breakdown-related genes BAM1, BAM3 and BAM4 was upregulated in the OX32.8 plants, whereas the expression of BAM6 and GWD3 was downregulated in the SRDX32.6 plants (Supplementary Fig. S4A). Taken together, these data indicate that ANAC032 represses photosynthesis and reduces sugar accumulation in plants. Fig. 2 ANAC032 regulates soluble sugars and starch levels. Ten-daysold Col-0, OX32 and SRDX32 plants grown in solid 1/2-strength MS media. (A) Sugar contents. (B) Starch contents. The error bars represent the SE, and the asterisks indicate significant differences from the control (Student’s t-test, P < 0.01). Studies have shown that carbon starvation results in autophagy (Ishizaki et al. 2006, Izumi et al. 2013). Thus, we analyzed the expression of autophagy-related genes (ATGs). The gene expression of eight ATGs, ATG8E, ATG8F, ATG8G, ATG8H, ATG13, ATG18B, ATG18C and ATG18D, was upregulated in the OX32.8 plants, whereas the expression of ATG6, ATG8H and ATG18G was downregulated in the SRDX32.6 plants (Fig. 3C). We also found that the expression of several autophagy and ER stress-related genes, WRKY33, metacaspase 5 (MC5), AATP1, At1G69325 and At2G27140, was upregulated in the OX32.8 plants but downregulated in the SRDX32.6 plants (Fig. 3C). ANAC032 regulates the carbon metabolism pathway To further confirm that ANAC032 induces the carbon starvation response, we examined genes associated with the glycolysis pathway, tricarboxylic acid (TCA) cycle and glyoxylic acid cycle enzymes (Fig. 4). In the glycolysis pathway, the expression of 1001 L. Sun et al. | ANAC032 is a regulator of carbon/energy status Table 1 Metabolite contents in 8-days-old Col-0, OX32.8 and SRDX32.6 seedlings grown on 1/2-strength MS media Metabolites Col-0 OX32.8 SRDX32.6 4.5 ± 0.3 3.7 ± 0.2 6.1 ± 0.5a FW) 2 ± 0.19 2.3 ± 0.3 2.5 ± 0.4a Sucrose (mg g1 FW) 5.5 ± 0.2 5.0 ± 0.3a 6.4 ± 0.4a Maltose (mg g1 FW) 80.1 ± 2.9 62.4 ± 4.4a 98 ± 3.25a FW) 100.3 ± 5.1 a 123 ± 4.8 127.5 ± 4.1a Raffinose (mg g1 FW) 156 ± 7.6 115 ± 4.5a 131 ± 9.1a 3.5 ± 0.2 a 3 ± 0.2 3.7 ± 0.3 54.6 ± 2.4 60 ± 2 89 ± 4.3a 1.74 ± 0.22 2.1 ± 0.14 2.7 ± 0.1a 2.2 ± 0.23 a 3 ± 0.19 5.2 ± 0.21a 51.2 ± 3.4 64 ± 4.6a 70 ± 6a FW) 2.3 ± 0.019 2.7 ± 0.016 2.78 ± 0.04a Succinate (mg g1 FW) 1.6 ± 0.06 2.34 ± 0.04a 2.89 ± 0.12a Oxaloacetate (mg g1 FW) 8.7 ± 0.05 10.1 ± 0.1a 8.89 ± 0.29 6.7 ± 0.03 6.4 ± 0.03 8.6 ± 0.07a 7.3 ± 0.14 9.5 ± 0.23a 11 ± 0.5a 57.1 ± 6.9 59.2 ± 8.7 73.3 ± 4.3a 1,985.8 ± 234.3 1,843.9 ± 110.5 1,829.7 ± 159.5 1 Fructose (mg g Mannose (mg g 1 1 Trehalose (mg g 1 Malate (mg g FW) FW) Shikimate (mg g1 FW) 1 Citrate (mg g FW) Fumarate (mg g1 FW) Aconitate (mg g 1 a-Ketoglutarate (mg g 1 Glyoxylate (mg g1 FW) 1 Pyruvate (mg g Asp (mg g1 FW) FW) FW) a 1 FW) 1,923.2 ± 167 1,758 ± 93 2,427.8 ± 88.9a 1 FW) 643.5 ± 68 634.9 ± 52.1 602.2 ± 28 1 FW) 558.2 ± 59 544.3 ± 68.5 551.2 ± 13.7 His (mg g1 FW) 218 ± 21 208 ± 24.7 193.9 ± 10.1 FW) 730.3 ± 76.9 644.9 ± 57 866 ± 36.4a Thr (mg g1 FW) 586.5 ± 64.9 564.8 ± 52.1 553.4 ± 23.3 Ala (mg g1 FW) 641.9 ± 71.5 611.1 ± 57.5 590.6 ± 28.5 Pro (mg g1 FW) 451.8 ± 27.9 428.3 ± 11.2 492.1 ± 13.4a a 410.1 ± 6.1a Glu (mg g Ser (mg g Gly (mg g Arg (mg g 1 1 FW) 361 ± 19 332.1 ± 13 Val (mg g1 FW) 508.5 ± 55.5 453.7 ± 30.2 419.6 ± 23.1a Tyr (mg g Met (mg g1 FW) 13.8 ± 2.7 15 ± 5.9 10.3 ± 2 Cys (mg g1 FW) 49.3 ± 3.4 34 ± 7a 69.4 ± 1.7a Ile (mg g1 FW) 363.3 ± 37 338.6 ± 27.6 318.6 ± 19.7 Leu (mg g1 FW) 902.6 ± 82.8 778.3 ± 62.2a 860 ± 34.3 476.2 ± 23.2 469.8 ± 40.8 456.4 ± 37.3 710.3 ± 35.2 a 690.1 ± 21.4 1 Phe (mg g 1 Lys (mg g FW) FW) 654.3 ± 24.1 The error bars represent the SEs. a Significant differences from the Col-0 controls (Student’s t-test, P < 0.05). five glucose-6-phosphate dehydrogenase genes (G6PD1, G6PD2, G6PD4, G6PD5 and G6PD6), a phosphofructokinase gene (PFK3), two glyceraldehyde-3-phosphate dehydrogenase genes (GAPCP-1 and GAPCP-2), a phosphoenolpyruvate enolase gene (ENOC), a pyruvate kinase gene (PKp3) and three pyruvate dehydrogenase complex subunit genes (MAB1, IAR4 and LTA3) was upregulated in the OX32.8 plants, whereas the expression of G6PD1, G6PD5, G6PD6, PFK4, PKp2, PKp3, MAB1, IAR4 and LTA3 was downregulated in the SRDX32 plants. In the TCA cycle and glyoxylic acid cycle, the expression of citrate synthase (CSY2), aconitase (ACO2), isocitrate dehydrogenase (ICDH), isocitrate lyase (ICL) and two succinate dehydrogenase subunit (SDH1-1 and SDH2-1) genes was upregulated in the OX32.8 plants, whereas the expression of ACO2, ICDH, SDH1-1, SDH2-1, a 1002 fumarate hydratase gene (FUM2) and two malate dehydrogenase genes (c-NAD-MDH3 and PMDH2) was downregulated in the SRDX32.6 plants (Fig. 4). We also examined the contents of several primary metabolites and found that ANAC032 alters the levels of glycolysis and TCA cycle intermediates (Fig. 4 and Table 1). Compared with the Col-0 and OX32.8 plants, the SRDX32.6 plants accumulate higher levels of pyruvate, malate, shikimate, citrate, fumarate, aconitate, succinate, glyoxylate and a-ketoglutarate. These data suggest that ANAC032 reprograms sugar metabolism processes. However, we also found that both the OX32.8 and SRDX32.6 lines showed higher levels of citrate, fumarate, succinate and glyoxylate than the Col-0 plants. These results are discussed below. Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 FW) Glucose (mmol g 1 Plant Cell Physiol. 60(5): 999–1010 (2019) doi:10.1093/pcp/pcz015 Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 Fig. 3 (A, B) ANAC032 induces carbon starvation responses. Number of expressed (A) carbon fixation-repressed genes and (B) carbon fixationinduced genes in OX32 plants. FC, fold change. (C, D) ANAC032 induces autophagy and ER stress responses. (C) The heat maps indicate a log2 FC in the expression of genes involved in autophagy and ER stress responses relative to that of Col-0. ANAC032 regulates primary nitrogen assimilation and amino acid catabolism The above results indicate that ANAC032 triggers sugar catabolism and induces autophagy. Sugar starvation-induced autophagy affects nitrogen assimilation and contributes to amino acid catabolism (Ishizaki et al. 2006, Izumi et al. 2013, Garapati et al. 2015a). Therefore, we analyzed the possible roles of ANAC032 in the modulation of primary nitrogen assimilation and amino acid catabolism (Fig. 4). In the primary nitrogen assimilation pathway, the overexpression of ANAC032 upregulates the gene expression of two nitrate reductases (NIA1 and NIA2), three glutamine synthetases (GLN1;1, GLN1;3 and GLN1;4), a glutamate synthase (GLU1), an aspartate aminotransferase (ASP3), two glutamine synthetases (GLN1;1 and GLN1;3), two glutamate dehydrogenases (GDH2 and GDH3) and a glutamate decarboxylase (GAD4), whereas the SRDX32 line exhibited reduced gene expression of NIA2, nitrite reductase (NIR1), two glutamate synthases (GLT1 and GLU1), GLN1;4 and GAD2. These data indicated that overexpression of ANAC032 promotes primary nitrogen assimilation. Aminotransferases redirect nitrogen resources into different pathways (Brosnan 2000). The overexpression of ANAC032 upregulates the expression of the genes encoding alanine aminotransferase (AGT3), two branched-chain amino acid aminotransferases (BCAT-1 and BCAT-2) and tyrosine aminotransferase (TAT7) (Fig. 5). The analysis of the expression of amino acid catabolismrelated genes revealed that the expression of genes involved in the branched-chain amino acid catabolism pathway and the Lys, Tyr, Trp, Met and Cys catabolism pathways is upregulated in the OX32.8 plants (Fig. 5); these genes include branched-chain amino acid transaminases (BCATs), branched- chain -keto acid dehydrogenase E1 (DIN4), electron transfer flavoprotein -subunit (ETFBETA), aldehyde dehydrogenases (ALDHs), acyl-CoA oxidase (ACX5), 4-hydroxyphenylpyruvate dioxygenase (PDS1) and homogentisate 1,2-dioxygenase (HGO) (Fig. 5). These data indicated that overexpression of ANAC032 promotes amino acid catabolic processes. Amino acid catabolism requires coordination with amino acid transport and carbohydrate metabolism. Our transcriptome data analysis also revealed that overexpression of ANAC032 upregulates the expression of several nodulin MtN21-like transporter family proteins (UMAMIT10/14/20/ 28/29/30//45), which are involved in amino acid transport, whereas the SRDX32.6 plants exhibited reduced expression of UMAMIT14/20/29/30) (Supplementary Fig. S4A). We also analyzed the free amino acid contents and observed that ANAC032 activation affects the free amino acid levels in seedlings (Table 1). The overexpression of ANAC032 reduced the levels of Tyr, Cys, Leu and Lys, whereas the SRDX32.6 plants presented increased levels of Tyr, Cys, Glu, Arg and Pro. ANAC032 regulates trehalose metabolism by directly regulating TRE1 expression The above results indicate that ANAC032 triggers carbon and nitrogen catabolism and induces carbon starvation responses in plants. A previous study indicated that ATAF1, which is homologous to ANAC032, triggers sucrose starvation-type changes by regulating TRE1 expression (Garapati et al. 2015a). In plants, trehalose metabolism is an important regulatory pathway that regulates carbon use and energy status. Thus, we speculated whether ANAC032 affects sugar metabolism by directly regulating trehalose metabolism in addition to inhibiting photosynthesis to induce carbon starvation responses. Seven of the 11 1003 L. Sun et al. | ANAC032 is a regulator of carbon/energy status trehalose-6-phosphate synthase (TPS) genes, 7 of the 10 trehalose-phosphate phosphatase (TPP) genes, and the single gene TRE1, which catalyzes trehalose hydrolysis to form glucose, were differentially expressed in the OX32 and SRDX32 lines. The expression of four TPS genes (TPS8/9/10/11), three TPP genes (TPPD, TPPE and TPPJ), and TRE1 was upregulated in the OX32.8 line, whereas the expression of four TPS genes (TPS1/ 6/7/11) and four TPP genes (TPPA, TPPE, TPPF and TPPJ) was downregulated in the SRDX32.6 line (Fig. 6A). We also measured the contents of trehalose and trehalose-6-phosphate (T6P) and found that the contents of trehalose and T6P were 1004 slightly (but statistically significantly) reduced in the OX32.8 plants, whereas, the T6P content was significantly increased in the SRDX32.6 plants (Fig. 6B, C). Trehalose inhibits plant growth in high concentration (Garapati et al. 2015a). Loss-of-function tre1 mutant is hypersensitive to exogenous trehalose, whereas TRE1 overexpression plants show increased tolerance (Van Houtte et al. 2013, Garapati et al. 2015a). We thus examined the primary root (PR) growth in the OX32 and SRDX32 lines (Supplementary Fig. S5). Exogenous trehalose markedly represses PR growth, and the inhibitory effects are less in OX32 lines, whereas are Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 Fig. 4 ANAC032 regulates the gene expression profiles and intermediate levels in glycolysis, the TCA cycle, the glyoxylic acid cycle and primary nitrogen metabolism. The heat maps indicate the log2 fold change in the expression of genes involved in glycolysis, the TCA cycle, the glyoxylic acid cycle and primary nitrogen metabolism relative to that in Col-0. Metabolites with higher or lower abundances in the OX32.8 (left) or SRDX32.6 (right) plants compared with those in the Col-0 plants are represented by red (higher) and blue (lower) circles within the boxes. The gray circles represent metabolites whose abundance was unchanged. Plant Cell Physiol. 60(5): 999–1010 (2019) doi:10.1093/pcp/pcz015 Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 Fig. 5 ANAC032 regulates amino acid catabolism processes. The heat maps indicate a log2 fold change in the expression of genes involved in the catabolism of Leu, Val, Ile, Lys, Trp, Cys, Met and Tyr relative to that of Col-0. aggravated in SRDX32.6 line (Supplementary Fig. S5). Taken together, these data indicate that ANAC032 regulates trehalose metabolism. TRE1 plays a critical role in the regulation of trehalose metabolism; thus, subsequently, we examined whether ANAC032 directly regulates the expression of the TRE1 gene using ChIPPCR. By searching for the T(A/C/G)CGT(A/G) and G(A/T/C/ G)G(A/G)G(A/G)G(A/G) elements of the NAC binding motif (Shamimuzzaman and Vodkin 2013) in the promoter of the TRE1 gene, we designed primers for ChIP-PCR assays. ANAC032 binding was enriched in the promoter region of TRE1 (Fig. 6D). To further confirm these results, we also examined the function of ANAC032 in promoting the transcription of TRE1 by analyzing ANAC032-mediated transcriptional activity using a protoplast cotransfection luciferase reporter assay. The results showed that ANAC032 induces transcription via the promoter of TRE1 (Fig. 6E). Taken together, these data indicate that ANAC032 directly regulates the expression of TRE1. Discussion Environmental cues induce carbon starvation responses in plants (Lin and Wu 2004, Lu et al. 2006, Usadel et al. 2008, Wu et al. 2009, Garapati et al. 2015b). Carbon starvation responses trigger sugar and amino acid catabolism, thereby maintaining the energy supply; therefore, carbon starvation response processes in plants maintain the survival of plants under environmental stresses. Garapati et al. (2015a) reported that ATAF1, which is homologous to ANAC032, is markedly induced by carbon starvation and leads to the reprogramming of the transcriptome and metabolome in response to carbon starvation by regulating TRE1 expression. In this study, our results showed that ANAC032 promotes carbon and nitrogen catabolism through the regulation of trehalose metabolism (and direct regulation of TRE1 expression); however, ANAC032 also induces carbon starvation by repressing photosynthesis and finally modulating stress responses. Our transcriptome data indicated that the overexpression of ANAC032 leads to the downregulation of photosynthesisrelated gene expression. Repressed photosynthesis promotes ROS accumulation in chloroplasts and, thus, regulates plant immune responses (Allu et al. 2016). Based on the results of a previous study (Allu et al. 2016) and our results, ANAC032 leads to photosynthesis inhibition and subsequent ROS accumulation, along with the direct regulation of pathogen-responsive gene expression, to coordinately regulate plant defense. Studies have shown that ANAC032 regulates pathogen defenses in plants (Allu et al. 2016) and represses anthocyanin production (Mahmood et al. 2016b). Similar to the results of Allu et al. (2016), our transcriptome data analysis revealed that ANAC032 regulates the expression of pathogen response genes (Supplementary Table S5). Moreover, consistent with the results reported by Mahmood et al. (2016b), our transcriptome data analysis revealed that the SRDX32 chimeric repressor plants 1005 L. Sun et al. | ANAC032 is a regulator of carbon/energy status presented elevated expression of several anthocyanin biosynthesis-related genes (DFR, TT8, LDOX, UF3GT, TT5/CHI, F3H/TT6, F30 H/TT7 and FLS1), whereas MYBL2, which is a negative regulator of anthocyanin biosynthesis, was downregulated in the SRDX32 line (Supplementary Fig. S6). In addition, the KEGG functional enrichment analysis revealed that the DEGs in the SRDX32 line were enriched in the anthocyanin biosynthesis pathway. Several studies have revealed a positive correlation between abiotic stress tolerance and anthocyanin production in plants, which is attributable to the ROS-scavenging function of anthocyanins (Li et al. 2017). Indeed, SRDX32 plants are more tolerant to abiotic stress than OX32 plants (Mahmood et al. 2016a). Previous studies have shown that ANAC032 induces developmental leaf senescence by modulating ROS production 1006 (Mahmood et al. 2016a). ANAC032 also induces the expression of several SAGs (Mahmood et al. 2016a). Similar to those results, our transcriptome data analysis revealed that the expression of 42 SAGs (81%, log2-fold change > 1) was upregulated in the ANAC032-overexpressing lines compared with that in the Col-0 controls, even in 8-day-old seedlings (Supplementary Fig. S7 and Supplementary Table S6). Additional studies are needed to elucidate whether ANAC032 regulates senescence by directly regulating the expression of SAGs. Taken together, these results indicate that our transcriptomic data are reliable. ANAC032 also induces the expression of autophagy and ER stress-related genes. ER stress response regulates both abiotic stress and biotic stress tolerance in plants (Yang et al. 2014, Cho and Kanehara 2017). Several studies have demonstrated that Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 Fig. 6 ANAC032 directly regulates the expression of the TRE1 gene. (A) Expression of trehalose metabolism-related genes in 8-day-old OX32.8, SRDX32.1 and SRDX32.6 plants grown on solid 1/2-strength MS media. The heat maps indicate the log2 fold change gene expression relative to that of Col-0. (B, C) Levels of trehalose (B) and T6P (C) in 8-day-old Col-0, OX32.8 and SRDX32.6 plants grown in solid 1/2-strength MS media. (D) ANAC032 binds to the promoter region of the TRE1 gene. A schematic of the primer design for the TRE1 gene is shown above the panel. The black line represents the promoter region, and the square represents the gene-coding region. The red lines with numbers denote the locations of amplicons amplified via ChIP-PCR. Anti-GFP antibodies were used to precipitate ANAC032-GFP. The asterisks indicate significant differences from the GFP-free control (Student’s t-test, P < 0.01). (E) Transient dual-luciferase reporter assays showing that ANAC032 promotes transcription via the TRE1 promoter. The ANAC032 promoter (1.5 kb) was fused to the luciferase reporter gene and transfected either alone or in combination with the 35S:ANAC032 plasmid. The experiment was performed for six biological replicates. The error bars represent the SEs, and the asterisks indicate significant differences from the control (Student’s t-test, P < 0.01). Plant Cell Physiol. 60(5): 999–1010 (2019) doi:10.1093/pcp/pcz015 of glycolytic enzymes increases metabolites, which could allosterically inhibit the rate-limiting step in glycolysis, resulting in no changes in the rate of glycolysis (Schaaff et al. 1989, Koebmann et al. 2002). This explanation is particularly applicable to the TCA cycle during which citrate also inhibits the ratelimiting step in glycolysis, resulting in a decrease or no change in glycolysis, which also affects the rate of TCA (Williamson and Jones 1964). Therefore, the changes in metabolism were derived from both transcriptional levels and the feedback regulation of cellular metabolites. Carbon starvation affects trehalose metabolism (Lunn et al. 2006, Ma et al. 2011). The SnRK1-bZIP11-T6P module regulates carbon starvation responses by integrating metabolic signals to regulate plant growth (Paul 2008, Smeekens et al. 2010). ANAC032 triggers sugar starvation responses and reduces the levels of trehalose and T6P. Changes in trehalose metabolism modulate carbon use and energy status, although the underlying molecular mechanism remains largely unclear (Ma et al. 2011, Garapati et al. 2015a). TPSs, TPPs and TRE1 regulate trehalose metabolism in plants (Ma et al. 2011, Vandesteene et al. 2012). Our study indicates that ANAC032 regulates the expression of the TPSs, TPPs and TRE1 genes. However, the function of most TPS genes is still unknown, and TPS5-11 do not appear to have catalytic (TPS or TPP) activity (Ramon et al. 2009). TRE1 encodes trehalase and plays a critical role in modulating trehalose metabolism. The results of our ChIP-PCR and protoplast cotransfection luciferase reporter assays indicated that similar to ATAF1, the TRE1 promoter is also a direct target of ANAC032, suggesting the direct regulation of TRE1 expression by both ATAF1 and ANAC032. In this study, the expression of DEG in OX32.8 was contrary to those in SRDX32.6 plants. However, the expression of most of DEG in OX32 plants that involved in carbon/ nitrogen metabolism were less affected in SRDX32.1 plants. These data were consistent with the observed results that SRDX32.6 plants show stronger phenotypes than SRDX32.1 plants. In summary, our data indicate that ANAC032 regulates the carbon starvation response by repressing photosynthesis via the downregulation of photosynthesis-related gene expression and the reprogramming of carbon and nitrogen metabolism by modulating trehalose metabolism and that ANAC032 and ATAF1 are functionally redundant in the process. Changes in sugar and amino acid catabolism increase energy supplies, thus maintaining plant survival under stresses. The plant response to carbon starvation is a complex process. Our study indicated that ANAC032 plays a critical role in these physiological processes (Supplementary Fig. S8). Future studies elucidating whether other regulators (such as ATAF1 and the group S1 basic leucine zipper TFs) of carbon starvation responses also regulate photosynthesis and the underlying molecular mechanisms of these regulators on the transcriptional regulation of photosynthesis will enable a broader understanding of the mechanisms underlying the intricate regulatory networks that link carbon starvation and defense and stress responses. Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 ANAC032 regulates the responses of plants to salt and drought stress and P. syringae infection (Allu et al. 2016, Mahmood et al. 2016a, Mahmood et al. 2016b). Future studies examining the interplay between ANAC032 and ER stress-related TFs will enable a better understanding of ANAC032-mediated defense and stress responses. A previous study showed that carbon starvation markedly induces ANAC032 expression (Bläsing et al. 2005). Here, by comparing the expression profile of ANAC032-regulated genes with that of carbon fixation-regulated genes (Bläsing et al. 2005), we found that the ANAC032-induced changes in the transcriptome and metabolome resemble a carbon-limitation state. The SRDX32 seedlings exhibited increased contents of soluble sugars (glucose, fructose and sucrose) and starch, whereas the OX32 seedlings exhibited reduced accumulation of glucose, sucrose and starch, supporting that ANAC032 induces carbon starvation responses in plants. The levels of most of the tested intermediates of glycolysis and the TCA cycle were higher in the ANAC032-SRDX plants than in the Col-0 and ANAC032-overexpressing plants, supporting that ANAC032 induces a sugar starvation response. These data also indicate that in addition to ANAC032, a redundant function exist in the SNAC-A subfamily of TFs to regulate primary carbon and nitrogen metabolism in plants. A comparison of the transcriptome data indicated that the expression of most of the DEGs that encode glycolysis and TCA cycle enzymes was upregulated in the ANAC032-overexpressing plants. These data suggested that ANAC032 increases sugar catabolism and energy supply processes. Our results showed that ANAC032 regulates primary nitrogen assimilation and amino acid catabolism by inducing the expression of primary nitrogen assimilation-related genes, amino acid catabolism-related genes (especially branchedchain amino acid catabolism-related genes) and amino acid transport-related UMAMIT genes. Hartmann et al. (2015) found that the SnRK1-bZIP1/53 module reprograms primary carbon and nitrogen metabolism by modulating branchedchain amino acid catabolism, highlighting the important roles of branched-chain amino acid catabolism in the modulation of primary carbon and nitrogen metabolism. Previous studies have indicated that autophagy could affect nitrogen assimilation and promote amino acid catabolism in response to abiotic stress (Izumi et al. 2013, Garapati et al. 2015a). Indeed, we observed that the expression of ATGs is also induced in the OX32 plants. These data support previous reports that carbon starvation both induces amino acid catabolism and causes the provision of alternative respiratory substrates to compensate for carbon starvation status for cellular energy homoeostasis (Ishizaki et al. 2006, Izumi et al. 2013, Garapati et al. 2015a). The metabolite analysis also revealed that several metabolites were increased in both the OX32 and SRDX32 plants. It is a bit strangely because the two plants should show opposite effects. The metabolic enzymes are highly regulated by cofactors and other cellular metabolites. For instance, the overexpression 1007 L. Sun et al. | ANAC032 is a regulator of carbon/energy status Materials and Methods Plant materials, growth conditions and phenotypic analysis Constructs With respect to ANAC032pro:GUS, 1.5-kb promoter regions were inserted via EcoRI/BamHI sites into pCAMBIA1381 plant expression vectors. For transient expression of ANAC032 in protoplasts, 35S-driven ANAC032 constructs were generated. The ANAC032 coding sequence was fused into the EcoRI/HindIII open reading frame (ORF) in a pGreenII 62-SK vector (Hellens et al. 2005). The promoter of the TRE1 (At4g24040) gene was fused to the luciferase gene via the HindIII/BamHI sites in a pGreenII 0800-LUC vector (Hellens et al. 2005). The fragments of the promoters or ORF were amplified from Arabidopsis Col-0 genomic DNA or cDNA, respectively. The primers used are listed in Supplementary Table S1. Measurement of chlorophyll fluorescence The chlorophyll fluorescence parameters were measured using an LI-6800 (LI-COR, America). The leaves of 5-week-old soilgrown plants were exposed to dark adaptation for 8 h, and then, F0 (minimum fluorescence) and Fm (maximum fluorescence yield) were measured under weak light conditions. The dark-adapted plants were exposed to 120 mmol m2 s1 light intensity for adequate light adaptation. For the induction kinetics measurements, the activated light intensity was set to 90 mmol m2 s1. The following parameters were recorded: F00 (minimum fluorescence yield of light adapted leaves), Fm0 (maximum fluorescence yield of light adapted leaves), Fs (fluorescence in a stable state) and PSII (effective quantum yield of PSII). The optimal photochemical efficiency of PSII in the dark (Fv/Fm) was calculated as (Fm F0)/Fm. The PQ redox state of PSII (1 qL) was calculated as (F00 /Fs)(Fm0 Fs)/(Fm0 F00 ) (Su et al. 2018). The experiments were repeated three times with 6–8 leaves in each replicate. Quantification of chlorophyll contents, soluble sugars, starch, free amino acids and primary metabolites Eight-day-old agar-grown seedlings were harvested for the determination of the chlorophyll contents and metabolite 1008 Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 The seeds of Col-0, the ANAC032 overexpression lines OX32.6 and OX32.8, and the chimeric repressor of ANAC032 lines SRDX32.1 and SRDX32.6 were surface sterilized and sown on 1/2-strength Murashige and Skoog (MS) medium (Sigma) consisting of 1% sucrose (pH 5.75). Using a qRT-PCR analysis, compared to Col-0 plants, the OX32.6 and OX32.8 lines have been shown to more highly express the transgene by approximately 107- and 104-fold, respectively, and the SRDX32.1 and SRDX32.6 line have also been shown to express the chimeric construct at higher levels than Col-0 plants (Mahmood et al. 2016a). For the trehalose (TrE) treatment, 30 mM TrE were added to the 1/2-strength MS media. The seedlings were grown in a growth chamber maintained at 22 C and maintained under a 16/8 h light/dark photoperiod under 50 mmol m2 s1 light after 2 d of stratification at 4 C. analysis. The samples were collected after 8 h of light. The total chlorophyll was extracted from the seedlings with 80% (v/v) acetone for 8 h and then measured based on A646 and A663 readings (Hanfrey et al. 1996). Seedlings (100 mg) were ground in 80% ethanol and then extracted twice at 80 C. The resulting supernatants were used to measure the soluble sugars in accordance with the methods of Eimert et al. (1995). The pellets were dried, and the starch contents were measured as described by Eimert et al. (1995). In vivo starch production was monitored via Lugol staining as described by Garapati et al. (2015a). The trehalose content was determined with a trehalose assay kit (Cominbio, China) according to the manufacturer’s instructions. The trehalose-6phosphate (T6P) contents were determined in chloroform/ methanol extracts using high-performance anion-exchange liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) as described by Figueroa et al. (2016). The free amino acid contents were determined using High Performance Liquid Chromatography (HPLC) as described by Zhang et al. (2010) with minor modifications. Briefly, frozen tissue (0.5 g) was ground in 1 ml of 20 mM cold HCl. Norleucine was used as an internal standard for measuring recovery. After derivatization, the mixture was transferred to a 100 ml glass insert in an amber vial and then was analyzed by HPLC (Rigol L3000-system, Rigol, Beijing, China). The chromatographic conditions were as follows: column, Kromasil C18 (250 mm 4.6 mm, 5 mm); injection volume, 10 ml; flow rate and time, 1 ml min1 for 45 min; and column temperature, 40 C. The soluble sugars and primary metabolite contents were determined using HPLC according to the methods described by Cruz et al. (2008) with minor modifications. Briefly, for the determination of malate, shikimate, citrate, fumarate and aconitate, the freeze-dried seedlings (100 mg) were ground in H2O; after sonication, the supernatants were filtered through 0.45-mm nylon filters and then analyzed via HPLC (Agilent 1100 system, Agilent Technologies). The chromatographic conditions were as follows: column, Kromasil C18 (250 mm 4.6 mm, 5 mm); injection volume, 10 ml; flow rate and time, 0.8 ml min1 for 20 min; column temperature, 25 C; UV wavelength, 214 nm; and mobile phase, 1.56 g NaH2PO4 dissolved in 800 ml H2O supplemented with 16 ml methanol, pH 2.8. For the determination of oxaloacetate, a-ketoglutarate and glyoxylate, the freeze-dried seedlings (200 mg) were ground in 20 mM cold HCl (0.2 ml) and extracted for 1 h at 4 C. The supernatants were filtered through 0.45-mm nylon filters, and then, the pH was adjusted to 6.8–7.4 with NaOH and phenylhydrazine hydrochloride. After a 30-min incubation at 25 C, HPLC (Agilent 1100 system, Agilent Technologies) was performed to analyze the contents. The chromatographic conditions were as follows: column, Kromasil C18 (250 mm 4.6 mm, 5 mm); injection volume, 10 ml; flow rate and time, 1 ml min1 for 50 min; column temperature, 30 C; UV wavelength, 325 nm; and mobile phase, a mixture of 50 ml methanol and 950 ml ddH2O supplemented with 1.68 g K2HPO4 and 0.217 g KH2PO4. The experiments were repeated three times. Plant Cell Physiol. 60(5): 999–1010 (2019) doi:10.1093/pcp/pcz015 Chromatin immunoprecipitation (ChIP)-qPCR assay Transient dual-luciferase reporter assay Arabidopsis mesophyll protoplasts from 4-week-old plants were prepared and co-transfected with the pGreen II 0800LUC and pGreen II 62-SK constructs as described by Yoo et al. (2007). After culturing the protoplasts for 16 h under low light conditions, the firefly LUC and renilla luciferase (REN) activities were quantified with a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. The relative REN activity was used as an internal control. The experiments were repeated six times. The primers used are listed in Supplementary Table S7. Supplementary Data Supplementary data are available at PCP online. Funding The National Key Research and Development Program of China [2016YFC0501901]; the China National Natural Sciences Foundation [31772383]; Basic Research Program of Qinghai Province [2019-ZJ-7033]; and the Yunnan Province Foundation for academic leader [2014HB043]. Disclosures The authors have conflicts of interest to declare. We thank the Public Technology Service Center of the Xishuangbanna Tropical Botanical Garden of CAS for providing research facilities. References Allu, A.D., Brotman, Y., Xue, G.P. and Balazadeh, S. (2016) Transcription factor ANAC032 modulates JA/SA signalling in response to Pseudomonas syringae infection. EMBO Rep. 17: 1578–1589. Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55: 373–399. Bläsing, O.E., Gibon, Y., Günther, M., Höhne, M., Morcuende, R., Osuna, D., et al. 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Downloaded from https://academic.oup.com/pcp/article/60/5/999/5300154 by guest on 17 October 2023 Approximately 3 g of 35S:ANAC032-GFP transgenic seedlings were washed and ground in liquid nitrogen. The chromatin DNA was isolated and then sonicated. The chromatin solution (300 ml) was diluted to 3 ml using ChIP dilution buffer (containing 10% Triton, 1 mM EDTA, 167 mM NaCl and 16.7 mM Tris-Cl pH 8.0) and then equally divided into two other tubes. Then, 40 ml protein G-agarose beads (SigmaAldrich) were added to each tube and incubated for 1 h at 4 C. The solutions were transferred to two pre-prepared fresh tubes; one tube contained 10 ml anti-GFP antibody (Ab291, Abcam) at a 1:150 dilution, and the other tube was a negative control with no additions. Then, 50 ml protein G-agarose beads were added to the two tubes for immunoprecipitation, and the samples were incubated and rotated gently at 4 C overnight. 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