H2O2 negatively regulates aluminum resistance via oxidation and degradation of the transcription factor STOP1.

Aluminum (Al) stress triggers the accumulation of hydrogen peroxide (H2O2) in roots. However, whether H2O2 plays a regulatory role in aluminum resistance remains unclear. In this study, we show that H2O2 plays a crucial role in regulation of Al resistance, which is modulated by the mitochondrion-localized pentatricopeptide repeat protein REGULATION OF ALMT1 EXPRESSION 6 (RAE6). Mutation in RAE6 impairs the activity of complex I of the mitochondrial electron transport chain, resulting in the accumulation of H2O2 and increased sensitivity to Al. Our results suggest that higher H2O2 concentrations promote the oxidation of SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1), an essential transcription factor that promotes Al resistance, thereby promoting its degradation by enhancing the interaction between STOP1 and the F-box protein RAE1. Conversely, decreasing H2O2 levels or blocking the oxidation of STOP1 leads to greater STOP1 stability and increased Al resistance. Moreover, we show that the thioredoxin TRX1 interacts with STOP1 to catalyze its chemical reduction. Thus, our results highlight the importance of H2O2 in Al resistance and regulation of STOP1 stability in Arabidopsis (Arabidopsis thaliana).

Plant immunity suppression via PHR1-RALF-FERONIA shapes the root microbiome to alleviate phosphate starvation.

The microbiome plays an important role in shaping plant growth and immunity, but few plant genes and pathways impacting plant microbiome composition have been reported. In Arabidopsis thaliana, the phosphate starvation response (PSR) was recently found to modulate the root microbiome upon phosphate (Pi) starvation through the transcriptional regulator PHR1. Here, we report that A. thaliana PHR1 directly binds to the promoters of rapid alkalinization factor (RALF) genes, and activates their expression under phosphate-starvation conditions. RALFs in turn suppress complex formation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) receptor through FERONIA, a previously-identified PTI modulator that increases resistance to certain detrimental microorganisms. Suppression of immunity via the PHR1-RALF-FERONIA axis allows colonization by specialized root microbiota that help to alleviate phosphate starvation by upregulating the expression of PSR genes. These findings provide a new paradigm for coordination of host-microbe homeostasis through modulating plant innate immunity after environmental perturbations.

Multilayered synergistic regulation of phytoalexin biosynthesis by ethylene, jasmonate, and MAPK signaling pathways in Arabidopsis.

Camalexin, an indolic antimicrobial metabolite, is the major phytoalexin in Arabidopsis thaliana, and plays a crucial role in pathogen resistance. Our previous studies revealed that the Arabidopsis mitogen-activated protein kinases MPK3 and MPK6 positively regulate pathogen-induced camalexin biosynthesis via phosphoactivating the transcription factor WRKY33. Here, we report that the ethylene and jasmonate (JA) pathways act synergistically with the MPK3/MPK6-WRKY33 module at multiple levels to induce camalexin biosynthesis in Arabidopsis upon pathogen infection. The ETHYLENE RESPONSE FACTOR1 (ERF1) transcription factor integrates the ethylene and JA pathways to induce camalexin biosynthesis via directly upregulating camalexin biosynthetic genes. ERF1 also interacts with and depends on WRKY33 to upregulate camalexin biosynthetic genes, indicating that ERF1 and WRKY33 form transcriptional complexes to cooperatively activate camalexin biosynthetic genes, thereby mediating the synergy of ethylene/JA and MPK3/MPK6 signaling pathways to induce camalexin biosynthesis. Moreover, as an integrator of the ethylene and JA pathways, ERF1 also acts as a substrate of MPK3/MPK6, which phosphorylate ERF1 to increase its transactivation activity and therefore further cooperate with the ethylene/JA pathways to induce camalexin biosynthesis. Taken together, our data reveal the multilayered synergistic regulation of camalexin biosynthesis by ethylene, JA, and MPK3/MPK6 signaling pathways via ERF1 and WRKY33 transcription factors in Arabidopsis.

The ubiquitin-proteasome system regulates meiotic chromosome organization.

Meiotic crossover (CO) recombination is tightly regulated by chromosome architecture to ensure faithful chromosome segregation and to reshuffle alleles between parental chromosomes for genetic diversity of progeny. However, regulation of the meiotic chromosome loop/axis organization is poorly understood. Here, we identify a molecular pathway for axis length regulation. We show that the cohesin regulator Pds5 can interact with proteasomes. Meiosis-specific depletion of proteasomes and/or Pds5 results in a similarly shortened chromosome axis, suggesting proteasomes and Pds5 regulate axis length in the same pathway. Protein ubiquitination is accumulated in pds5 and proteasome mutants. Moreover, decreased chromosome axis length in these mutants can be largely rescued by decreasing ubiquitin availability and thus decreasing protein ubiquitination. Further investigation reveals that two ubiquitin E3 ligases, SCF (Skp­Cullin­F-box) and Ufd4, are involved in this Pds5­ubiquitin/proteasome pathway to cooperatively control chromosome axis length. These results support the hypothesis that ubiquitination of chromosome proteins results in a shortened chromosome axis, and cohesin­Pds5 recruits proteasomes onto chromosomes to regulate ubiquitination level and thus axis length. These findings reveal an unexpected role of the ubiquitin­proteasome system in meiosis and contribute to our knowledge of how Pds5 regulates meiotic chromosome organization. A conserved regulatory mechanism probably exists in higher eukaryotes.

A novel microbial community restructuring strategy for enhanced hydrogen production using multiple pretreatments and CSTR operation.

To achieve rapid enrichment of the targeted hydrogen-producing bacterial population and reconstruction of the microbial community in the biological hydrogen-producing reactor, the activated sludge underwent multiple pretreatments using micro-aeration, alkaline treatment, and heat treatment. The activated sludge obtained from the multiple pretreatments was inoculated into the continuous stirred tank reactor (CSTR) for continuous operations. The community structure alteration and hydrogen-producing capability of the activated sludge were analyzed throughout the operation of the reactor. We found that the primary phyla in the activated sludge population shifted to Proteobacteria, Firmicutes, and Bacteroidetes, which collectively accounted for 96.69% after undergoing several pretreatments. This suggests that the multiple pretreatments facilitated in achieving the selective enrichment of the fermentation hydrogen-producing microorganisms in the activated sludge. The CSTR start-up and continuous operation of the biological hydrogen production reactor resulted in the reactor entering a highly efficient hydrogen production stage at influent COD concentrations of 4000 mg/L and 5000 mg/L, with the highest hydrogen production rate reaching 8.19 L/d and 9.33 L/d, respectively. The main genus present during the efficient hydrogen production stage in the reactor was Ethanoligenens, accounting for up to 33% of the total population. Ethanoligenens exhibited autoaggregation capabilities and a superior capacity for hydrogen production, leading to its prevalence in the reactor and contribution to efficient hydrogen production. During high-efficiency hydrogen production, flora associated with hydrogen production exhibited up to 46.95% total relative abundance. In addition, redundancy analysis (RDA) indicated that effluent pH and COD influenced the distribution of the primary hydrogen-producing bacteria, including Ethanoligenens, Raoultella, and Pectinatus, as well as other low abundant hydrogen-producing bacteria in the activated sludge. The data indicates that the multiple pretreatments and reactor's operation has successfully enriched the hydrogen-producing genera and changed the community structure of microbial hydrogen production.

Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis.

Camalexin is a major phytoalexin that plays a crucial role in disease resistance in Arabidopsis (Arabidopsis thaliana). We previously characterized the regulation of camalexin biosynthesis by the mitogen-activated protein kinases MPK3 and MPK6 and their downstream transcription factor WRKY33. Here, we report that the pathogen-responsive CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5) and CPK6 also regulate camalexin biosynthesis in Arabidopsis. Chemically induced expression of constitutively active CPK5 or CPK6 variants was sufficient to induce camalexin biosynthesis in transgenic Arabidopsis plants. Consistently, the simultaneous mutation of CPK5 and CPK6 compromised camalexin production in Arabidopsis induced by the fungal pathogen Botrytis cinerea Moreover, we identified that WRKY33 functions downstream of CPK5/CPK6 to activate camalexin biosynthetic genes, thereby inducing camalexin biosynthesis. CPK5 and CPK6 interact with WRKY33 and phosphorylate its Thr-229 residue, leading to an increase in the DNA binding ability of WRKY33. By contrast, the MPK3/MPK6-mediated phosphorylation of WRKY33 on its N-terminal Ser residues enhances the transactivation activity of WRKY33. Furthermore, both gain- and loss-of-function genetic analyses demonstrated the cooperative regulation of camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6. Taken together, these findings indicate that WRKY33 functions as a convergent substrate of CPK5/CPK6 and MPK3/MPK6, which cooperatively regulate camalexin biosynthesis via the differential phospho-regulation of WRKY33 activity.

MPK3/6-induced degradation of ARR1/10/12 promotes salt tolerance in Arabidopsis.

Cytokinins are phytohormones that regulate plant development, growth, and responses to stress. In particular, cytokinin has been reported to negatively regulate plant adaptation to high salinity; however, the molecular mechanisms that counteract cytokinin signaling and enable salt tolerance are not fully understood. Here, we provide evidence that salt stress induces the degradation of the cytokinin signaling components Arabidopsis (Arabidopisis thaliana) response regulator 1 (ARR1), ARR10 and ARR12. Furthermore, the stress-activated mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ARR1/10/12 to promote their degradation in response to salt stress. As expected, salt tolerance is decreased in the mpk3/6 double mutant, but enhanced upon ectopic MPK3/MPK6 activation in an MKK5DD line. Importantly, salt hypersensitivity phenotypes of the mpk3/6 line were significantly alleviated by mutation of ARR1/12. The above results indicate that MPK3/6 enhance salt tolerance in part via their negative regulation of ARR1/10/12 protein stability. Thus, our work reveals a new molecular mechanism underlying salt-induced stress adaptation and the inhibition of plant growth, via enhanced degradation of cytokinin signaling components.

Acute and chronic effects of sublethal neonicotinoid thiacloprid to Asian honey bee (Apis cerana cerana).

Pesticide pollution is one of the most important factors for global bee declines. Despite many studies have revealed that the most important Chinese indigenous species，Apis cerana, is presenting a high risk on exposure to neonicotinoids, the toxicology information on Apis cerana remain limited. This study was aimed to determine the acute and chronic toxic effects of thiacloprid (IUPAC name: {(2Z)-3-[(6-Chloro-3-pyridinyl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide) on behavioral and physiological performance as well as genome-wide transcriptome in A. cerana. We found the 1/5 LC50 of thiacloprid significantly impaired learning and memory abilities after both acute and chronic exposure, nevertheless, has no effects on the sucrose responsiveness and phototaxis climbing ability of A. cerana. Moreover, activities of detoxification enzyme P450 monooxygenases and CarE were increased by short-term exposure to thiacloprid, while prolonged exposure caused suppression of CarE activity. Neither acute nor chronic exposure to thiacloprid altered honey bee AChE activities. To further study the potential defense molecular mechanisms in Asian honey bee under pesticide stress, we analyzed the transcriptomes of honeybees in response to thiacloprid stress. The transcriptomic profiles revealed consistent upregulation of immune- and stress-related genes by both acute or chronic treatments. Our results suggest that the chronic exposure to thiacloprid produced greater toxic effects than a single administration to A. cerana. Altogether, our study deepens the understanding of the toxicological characteristic of A. cerana against thiacloprid, and could be used to further investigate the complex molecular mechanisms in Asian honey bee under pesticide stress.

Comparative Transcriptome Analysis between Resistant and Susceptible Pakchoi Cultivars in Response to Downy Mildew.

Downy mildew caused by the obligate parasite Hyaloperonospora brassicae is a devastating disease for Brassica species. Infection of Hyaloperonospora brassicae often leads to yellow spots on leaves, which significantly impacts quality and yield of pakchoi. In the present study, we conducted a comparative transcriptome between the resistant and susceptible pakchoi cultivars in response to Hyaloperonospora brassicae infection. A total of 1073 disease-resistance-related differentially expressed genes were identified using a Venn diagram. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that these genes were mainly involved in plant-pathogen interaction, plant hormone signal transduction, and other photosynthesis-related metabolic processes. Analysis of the phytohormone content revealed that salicylic acid increased significantly in the resistant material after inoculation with Hyaloperonospora brassicae, whereas the contents of jasmonic acid, abscisic acid, and 1-aminocyclopropane-1-carboxylic acid decreased. Exogenous salicylic acid treatment also significantly upregulated Hyaloperonospora brassicae-induced genes, which further confirmed a crucial role of salicylic acid during pakchoi defense against Hyaloperonospora brassicae. Based on these findings, we suggest that the salicylic-acid-mediated signal transduction contributes to the resistance of pakchoi to downy mildew, and PAL1, ICS1, NPR1, PR1, PR5, WRKY70, WRKY33, CML43, CNGC9, and CDPK15 were involved in this responsive process. Our findings evidently contribute to revealing the molecular mechanism of pakchoi defense against Hyaloperonospora brassicae.

Cell type-specific proteomics uncovers a RAF15-SnRK2.6/OST1 kinase cascade in guard cells.

Multicellular organisms such as plants contain various cell types with specialized functions. Analyzing the characteristics of each cell type reveals specific cell functions and enhances our understanding of organization and function at the organismal level. Guard cells (GCs) are specialized epidermal cells that regulate the movement of the stomata and gaseous exchange, and provide a model genetic system for analyzing cell fate, signaling, and function. Several proteomics analyses of GC are available, but these are limited in depth. Here we used enzymatic isolation and flow cytometry to enrich GC and mesophyll cell protoplasts and perform in-depth proteomics in these two major cell types in Arabidopsis leaves. We identified approximately 3,000 proteins not previously found in the GC proteome and more than 600 proteins that may be specific to GC. The depth of our proteomics enabled us to uncover a guard cell-specific kinase cascade whereby Raf15 and Snf1-related kinase2.6 (SnRK2.6)/OST1(open stomata 1) mediate abscisic acid (ABA)-induced stomatal closure. RAF15 directly phosphorylated SnRK2.6/OST1 at the conserved Ser175 residue in its activation loop and was sufficient to reactivate the inactive form of SnRK2.6/OST1. ABA-triggered SnRK2.6/OST1 activation and stomatal closure was impaired in raf15 mutants. We also showed enrichment of enzymes and flavone metabolism in GC, and consistent, dramatic accumulation of flavone metabolites. Our study answers the long-standing question of how ABA activates SnRK2.6/OST1 in GCs and represents a resource potentially providing further insights into the molecular basis of GC and mesophyll cell development, metabolism, structure, and function.

Genome-wide identification and expression analysis reveals spinach brassinosteroid-signaling kinase (BSK) gene family functions in temperature stress response.

BACKGROUND: Brassinosteroid (BR)- signaling kinase (BSK) is a critical family of receptor-like cytoplasmic kinase for BR signal transduction, which plays important roles in plant development, immunity, and abiotic stress responses. Spinach (Spinacia oleracea) is cold- tolerant but heat- sensitive green leafy vegetable. A study on BSK family members and BSKs- mediated metabolic processes in spinach has not been performed. RESULTS: We identified and cloned seven SoBSKs in spinach. Phylogenetic and collinearity analyses suggested that SoBSKs had close relationship with dicotyledonous sugar beet (Beta vulgaris) rather than monocotyledons. The analyses of gene structure and conserved protein domain/ motif indicated that most SoBSKs were relative conserved, while SoBSK6 could be a truncated member. The prediction of post-translation modification (PTM) sites in SoBSKs implied their possible roles in signal transduction, redox regulation, and protein turnover of SoBSKs, especially the N-terminal myristoylation site was critical for BSK localization to cell periphery. Cis-acting elements for their responses to light, drought, temperature (heat and cold), and hormone distributed widely in the promoters of SoBSKs, implying the pivotal roles of SoBSKs in response to diverse abiotic stresses and phytohormone stimuli. Most SoBSKs were highly expressed in leaves, except for SoBSK7 in roots. Many SoBSKs were differentially regulated in spinach heat- sensitive variety Sp73 and heat- tolerant variety Sp75 under the treatments of heat, cold, as well as exogenous brassinolide (BL) and abscisic acid (ABA). The bsk134678 mutant Arabidopsis seedlings exhibited more heat tolerance than wild- type and SoBSK1- overexpressed seedlings. CONCLUSIONS: A comprehensive genome- wide analysis of the BSK gene family in spinach presented a global identification and functional prediction of SoBSKs. Seven SoBSKs had relatively- conserved gene structure and protein function domains. Except for SoBSK6, all the other SoBSKs had similar motifs and conserved PTM sites. Most SoBSKs participated in the responses to heat, cold, BR, and ABA. These findings paved the way for further functional analysis on BSK- mediated regulatory mechanisms in spinach development and stress response.

Myc-Miz1 signaling promotes self-renewal of leukemia stem cells by repressing Cebpα and Cebpδ.

c-Myc (Myc hereafter) is found to be deregulated and/or amplified in most acute myeloid leukemias (AMLs). Almost all AML cells are dependent upon Myc for their proliferation and survival. Thus, Myc has been proposed as a critical anti-AML target. Myc has Max-mediated transactivational and Myc-interacting zinc finger protein 1 (Miz1)-mediated transrepressional activities. The role of Myc-Max-mediated transactivation in the pathogenesis of AML has been well studied; however, the role of Myc-Miz1-mediated transrepression in AML is still somewhat obscure. Myc protein harboring a V394D mutation (MycV394D) is a mutant form of Myc that lacks transrepressional activity due to a defect in its ability to interact with Miz1. We found that, compared with Myc, the oncogenic function of MycV394D is significantly impaired. The AML/myeloproliferative disorder that develops in mice receiving MycV394D-transduced hematopoietic stem/progenitor cells (HSPCs) is significantly delayed compared with mice receiving Myc-transduced HSPCs. Using a murine MLL-AF9 AML model, we found that AML cells expressing MycV394D (intrinsic Myc deleted) are partially differentiated and show reductions in both colony-forming ability in vitro and leukemogenic capacity in vivo. The reduced frequency of leukemia stem cells (LSCs) among MycV394D-AML cells and their reduced leukemogenic capacity during serial transplantation suggest that Myc-Miz1 interaction is required for the self-renewal of LSCs. In addition, we found that MycV394D-AML cells are more sensitive to chemotherapy than are Myc-AML cells. Mechanistically, we found that Myc represses Miz1-mediated expression of CCAAT/enhancer-binding protein α (Cebpα) and Cebpδ, thus playing an important role in the pathogenesis of AML by maintaining the undifferentiated state and self-renewal capacity of LSCs.

Genome-Wide Identification of the A20/AN1 Zinc Finger Protein Family Genes in Ipomoea batatas and Its Two Relatives and Function Analysis of IbSAP16 in Salinity Tolerance.

Stress-associated protein (SAP) genes-encoding A20/AN1 zinc-finger domain-containing proteins-play pivotal roles in regulating stress responses, growth, and development in plants. They are considered suitable candidates to improve abiotic stress tolerance in plants. However, the SAP gene family in sweetpotato (Ipomoea batatas) and its relatives is yet to be investigated. In this study, 20 SAPs in sweetpotato, and 23 and 26 SAPs in its wild diploid relatives Ipomoea triloba and Ipomoea trifida were identified. The chromosome locations, gene structures, protein physiological properties, conserved domains, and phylogenetic relationships of these SAPs were analyzed systematically. Binding motif analysis of IbSAPs indicated that hormone and stress responsive cis-acting elements were distributed in their promoters. RT-qPCR or RNA-seq data revealed that the expression patterns of IbSAP, ItbSAP, and ItfSAP genes varied in different organs and responded to salinity, drought, or ABA (abscisic acid) treatments differently. Moreover, we found that IbSAP16 driven by the 35 S promoter conferred salinity tolerance in transgenic Arabidopsis. These results provided a genome-wide characterization of SAP genes in sweetpotato and its two relatives and suggested that IbSAP16 is involved in salinity stress responses. Our research laid the groundwork for studying SAP-mediated stress response mechanisms in sweetpotato.

Genome-wide identification of PTI1 family in Setaria italica and salinity-responsive functional analysis of SiPTI1-5.

BACKGROUND: PTI1 (Pto-interacting 1) protein kinase belongs to the receptor-like cytoplasmic kinase (RLCK) group of receptor-like protein kinases (RLK), but lack extracellular and transmembrane domains. PTI1 was first identified in tomato (Solanum lycopersicum) and named SlPTI1, which has been reported to interact with bacterial effector Pto, a serine/threonine protein kinase involved in plant resistance to bacterial disease. Briefly, the host PTI1 specifically recognizes and interacts with the bacterial effector AvrPto, which triggers hypersensitive cell death to inhibit the pathogen growth in the local infection site. Previous studies have demonstrated that PTI1 is associated with oxidative stress and hypersensitivity. RESULTS: We identified 12 putative PTI1 genes from the genome of foxtail millet (Setaria italica) in this study. Gene replication analysis indicated that both segmental replication events played an important role in the expansion of PTI1 gene family in foxtail millet. The PTI1 family members of model plants, i.e. S. italica, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), maize (Zea mays), S. lycopersicum, and soybean (Glycine max), were classified into six major categories according to the phylogenetic analysis, among which the PTI1 family members in foxtail millet showed higher degree of homology with those of rice and maize. The analysis of a complete set of SiPTI1 genes/proteins including classification, chromosomal location, orthologous relationships and duplication. The tissue expression characteristics revealed that SiPTI1 genes are mainly expressed in stems and leaves. Experimental qRT-PCR results demonstrated that 12 SiPTI1 genes were induced by multiple stresses. Subcellular localization visualized that all of foxtail millet SiPTI1s were localized to the plasma membrane. Additionally, heterologous expression of SiPTI1-5 in yeast and E. coli enhanced their tolerance to salt stress. CONCLUSIONS: Our results contribute to a more comprehensive understanding of the roles of PTI1 protein kinases and will be useful in prioritizing particular PTI1 for future functional validation studies in foxtail millet.

Establishment of an efficient Agrobacterium-mediated genetic transformation system in halophyte Puccinellia tenuiflora.

Alkaligrass (Puccinellia tenuiflora) is a monocotyledonous halophyte pasture, which has strong tolerance to saline-alkali, drought, and chilling stresses. We have reported a high-quality chromosome-level genome and stress-responsive proteomic results in P. tenuiflora. However, the gene/protein function investigations are still lacking, due to the absent of genetic transformation system in P. tenuiflora. In this study, we established a higher efficient Agrobacterium-mediated transformation for P. tenuiflora using calluses induced from seeds. Agrobacterium strain EHA105 harbors pANIC 6B vectors that contain GUS reporter gene and Hyg gene for screening. Ten mg·L-1 hygromycin was used for selecting transgenic calluses. The optimized condition of vacuum for 10 min, ultrasonication for 10 min, and then vacuum for 10 min was used for improvement of conversion efficiency. Besides, 300 mg·L-1 timentin was the optimum antibiotics in transformation. PCR amplification exhibited that GUS gene has been successfully integrated into the chromosome of P. tenuiflora. Histochemical GUS staining and qRT-PCR analysis indicated that GUS gene has stably expressed with ß-glucuronidase activity in transgene seedlings. All these demonstrated that we have successfully established an Agrobacterium-mediated transformation system of P. tenuiflora, which provides a good platform for further gene function analysis and lays a solid foundation for molecular breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-021-01247-8.

Exploring the diversity of plant proteome.

The tremendous functional, spatial, and temporal diversity of the plant proteome is regulated by multiple factors that continuously modify protein abundance, modifications, interactions, localization, and activity to meet the dynamic needs of plants. Dissecting the proteome complexity and its underlying genetic variation is attracting increasing research attention. Mass spectrometry (MS)-based proteomics has become a powerful approach in the global study of protein functions and their relationships on a systems level. Here, we review recent breakthroughs and strategies adopted to unravel the diversity of the proteome, with a specific focus on the methods used to analyze posttranslational modifications (PTMs), protein localization, and the organization of proteins into functional modules. We also consider PTM crosstalk and multiple PTMs temporally regulating the life cycle of proteins. Finally, we discuss recent quantitative studies using MS to measure protein turnover rates and examine future directions in the study of the plant proteome.

AtHB7/12 Regulate Root Growth in Response to Aluminum Stress.

Aluminum (Al) stress is a major limiting factor for plant growth and crop production in acid soils. At present, only a few transcription factors involved in the regulation of Al resistance have been characterized. Here, we used reversed genetic approach through phenotype analysis of overexpressors and mutants to demonstrate that AtHB7 and AtHB12, two HD-Zip I transcription factors, participate in Al resistance. In response to Al stress, AtHB7 and AtHB12 displayed different dynamic expression patterns. Although both AtHB7 and AtHB12 positively regulate root growth in the absence of Al stress, our results showed that AtHB7 antagonizes with AtHB12 to control root growth in response to Al stress. The athb7/12 double mutant displayed a wild-type phenotype under Al stress. Consistently, our physiological analysis showed that AtHB7 and AtHB12 oppositely regulate the capacity of cell wall to bind Al. Yeast two hybrid assays showed that AtHB7 and AtHB12 could form homo-dimers and hetero-dimers in vitro, suggesting the interaction between AtHB7 and AtHB12 in the regulation of root growth. The conclusion was that AtHB7 and AtHB12 oppositely regulate Al resistance by affecting Al accumulation in root cell wall.

NaCl-responsive ROS scavenging and energy supply in alkaligrass callus revealed from proteomic analysis.

BACKGROUND: Salinity has obvious effects on plant growth and crop productivity. The salinity-responsive mechanisms have been well-studied in differentiated organs (e.g., leaves, roots and stems), but not in unorganized cells such as callus. High-throughput quantitative proteomics approaches have been used to investigate callus development, somatic embryogenesis, organogenesis, and stress response in numbers of plant species. However, they have not been applied to callus from monocotyledonous halophyte alkaligrass (Puccinellia tenuifora). RESULTS: The alkaligrass callus growth, viability and membrane integrity were perturbed by 50 mM and 150 mM NaCl treatments. Callus cells accumulated the proline, soluble sugar and glycine betaine for the maintenance of osmotic homeostasis. Importantly, the activities of ROS scavenging enzymes (e.g., SOD, APX, POD, GPX, MDHAR and GR) and antioxidants (e.g., ASA, DHA and GSH) were induced by salinity. The abundance patterns of 55 salt-responsive proteins indicate that salt signal transduction, cytoskeleton, ROS scavenging, energy supply, gene expression, protein synthesis and processing, as well as other basic metabolic processes were altered in callus to cope with the stress. CONCLUSIONS: The undifferentiated callus exhibited unique salinity-responsive mechanisms for ROS scavenging and energy supply. Activation of the POD pathway and AsA-GSH cycle was universal in callus and differentiated organs, but salinity-induced SOD pathway and salinity-reduced CAT pathway in callus were different from those in leaves and roots. To cope with salinity, callus mainly relied on glycolysis, but not the TCA cycle, for energy supply.

Heat-Responsive Proteomics of a Heat-Sensitive Spinach Variety.

High temperatures seriously limit plant growth and productivity. Investigating heat-responsive molecular mechanisms is important for breeding heat-tolerant crops. In this study, heat-responsive mechanisms in leaves from a heat-sensitive spinach (Spinacia oleracea L.) variety Sp73 were investigated using two-dimensional gel electrophoresis (2DE)-based and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics approaches. In total, 257 heat-responsive proteins were identified in the spinach leaves. The abundance patterns of these proteins indicated that the photosynthesis process was inhibited, reactive oxygen species (ROS) scavenging pathways were initiated, and protein synthesis and turnover, carbohydrate and amino acid metabolism were promoted in the spinach Sp73 in response to high temperature. By comparing this with our previous results in the heat-tolerant spinach variety Sp75, we found that heat inhibited photosynthesis, as well as heat-enhanced ROS scavenging, stress defense pathways, carbohydrate and energy metabolism, and protein folding and turnover constituting a conservative strategy for spinach in response to heat stress. However, the heat-decreased biosynthesis of chlorophyll and carotenoid as well as soluble sugar content in the variety Sp73 was quite different from that in the variety Sp75, leading to a lower capability for photosynthetic adaptation and osmotic homeostasis in Sp73 under heat stress. Moreover, the heat-reduced activities of SOD and other heat-activated antioxidant enzymes in the heat-sensitive variety Sp73 were also different from the heat-tolerant variety Sp75, implying that the ROS scavenging strategy is critical for heat tolerance.

Proteomic discovery of H2O2 response in roots and functional characterization of PutGLP gene from alkaligrass.

MAIN CONCLUSION: Hydrogen peroxide-responsive pathways in roots of alkaligrass analyzed by proteomic studies and PutGLP enhance the plant tolerance to saline-, alkali- and cadmium-induced oxidative stresses. Oxidative stress adaptation is critical for plants in response to various stress environments. The halophyte alkaligrass (Puccinellia tenuiflora) is an outstanding pasture with strong tolerance to salt and alkali stresses. In this study, iTRAQ- and 2DE-based proteomics approaches, as well as qRT-PCR and molecular genetics, were employed to investigate H2O2-responsive mechanisms in alkaligrass roots. The evaluation of membrane integrity and reactive oxygen species (ROS)-scavenging systems, as well as abundance patterns of H2O2-responsive proteins/genes indicated that Ca2+-mediated kinase signaling pathways, ROS homeostasis, osmotic modulation, and transcriptional regulation were pivotal for oxidative adaptation in alkaligrass roots. Overexpressing a P. tenuiflora germin-like protein (PutGLP) gene in Arabidopsis seedlings revealed that the apoplastic PutGLP with activities of oxalate oxidase and superoxide dismutase was predominantly expressed in roots and played important roles in ROS scavenging in response to salinity-, alkali-, and CdCl2-induced oxidative stresses. The results provide insights into the fine-tuned redox-responsive networks in halophyte roots.