HISTONE DEACETYLASE 9 transduces heat signal in plant cells.

Heat stress limits plant growth, development, and crop yield, but how plant cells precisely sense and transduce heat stress signals remains elusive. Here, we identified a conserved heat stress response mechanism to elucidate how heat stress signal is transmitted from the cytoplasm into the nucleus for epigenetic modifiers. We demonstrate that HISTONE DEACETYLASE 9 (HDA9) transduces heat signals from the cytoplasm to the nucleus to play a positive regulatory role in heat responses in Arabidopsis. Heat specifically induces HDA9 accumulation in the nucleus. Under heat stress, the phosphatase PP2AB'ß directly interacts with and dephosphorylates HDA9 to protect HDA9 from 26S proteasome-mediated degradation, leading to the translocation of nonphosphorylated HDA9 to the nucleus. This heat-induced enrichment of HDA9 in the nucleus depends on the nucleoporin HOS1. In the nucleus, HDA9 binds and deacetylates the target genes related to signaling transduction and plant development to repress gene expression in a transcription factor YIN YANG 1-dependent and -independent manner, resulting in rebalance of plant development and heat response. Therefore, we uncover an HDA9-mediated positive regulatory module in the heat shock signal transduction pathway. More important, this cytoplasm-to-nucleus translocation of HDA9 in response to heat stress is conserved in wheat and rice, which confers the mechanism significant implication potential for crop breeding to cope with global climate warming.

Heat Shock Factor A1s are required for phytochrome-interacting factor 4-mediated thermomorphogenesis in Arabidopsis.

Thermomorphogenesis and the heat shock (HS) response are distinct thermal responses in plants that are regulated by PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and HEAT SHOCK FACTOR A1s (HSFA1s), respectively. Little is known about whether these responses are interconnected and whether they are activated by similar mechanisms. An analysis of transcriptome dynamics in response to warm temperature (28°C) treatment revealed that 30 min of exposure activated the expression of a subset of HSFA1 target genes in Arabidopsis thaliana. Meanwhile, a loss-of-function HSFA1 quadruple mutant (hsfa1-cq) was insensitive to warm temperature-induced hypocotyl growth. In hsfa1-cq plants grown at 28°C, the protein and transcript levels of PIF4 were greatly reduced, and the circadian rhythm of many thermomorphogenesis-related genes (including PIF4) was disturbed. Additionally, the nuclear localization of HSFA1s and the binding of HSFA1d to the PIF4 promoter increased following warm temperature exposure, whereas PIF4 overexpression in hsfa1-cq partially rescued the altered warm temperature-induced hypocotyl growth of the mutant. Taken together, these results suggest that HSFA1s are required for PIF4 accumulation at a warm temperature, and they establish a central role for HSFA1s in regulating both thermomorphogenesis and HS responses in Arabidopsis.

Heterologous expression of heat stress-responsive AtPLC9 confers heat tolerance in transgenic rice.

BACKGROUND: As global warming becomes increasingly severe, it is urgent that we enhance the heat tolerance of crops. We previously reported that Arabidopsis thaliana PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C9 (AtPLC9) promotes heat tolerance. RESULTS: In this study, we ectopically expressed AtPLC9 in rice to examine its potential to improve heat tolerance in this important crop. Whereas AtPLC9 did not improve rice tolerance to salt, drought or cold, transgenic rice did exhibit greater heat tolerance than the wild type. High-throughput RNA-seq revealed extensive and dynamic transcriptome reprofiling in transgenic plants after heat stress. Moreover, the expression of some transcription factors and calcium ion-related genes showed specific upregulation in transgenic rice after heat stress, which might contribute to the enhanced heat tolerance. CONCLUSIONS: This study provides preliminary guidance for using AtPLC9 to improve heat tolerance in cereal crops and, more broadly, highlights that heterologous transformation can assist with molecular breeding.

Arabidopsis cyclic nucleotide-gated channel 6 is negatively modulated by multiple calmodulin isoforms during heat shock.

An increased concentration of cytosolic Ca2+ is an early response of plant cells to heat shock. Arabidopsis cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca2+ influx and is activated by cAMP. However, it remains unclear how the Ca2+ conductivity of CNGC6 is negatively regulated under the elevated cytosolic Ca2+ concentration. In this study, Arabidopsis calmodulin isoforms CaM1/4, CaM2/3/5, CaM6, and CaM7 were found to bind to CNGC6 to varying degrees, and this binding was dependent on the presence of Ca2+ and IQ6, an atypical isoleucine-glutamine motif in CNGC6. Knockout of CaM2, CaM3, CaM5, and CaM7 genes led to a marked increase in plasma membrane inward Ca2+ current under heat shock conditions; however, knockout of CaM1, CaM4, and CaM6 genes had no significant effect on plasma membrane Ca2+ current. Moreover, the deletion of IQ6 from CNGC6 led to a marked increase in plasma membrane Ca2+ current under heat shock conditions. Taken together, the data suggest that CNGC6-mediated Ca2+ influx is likely to be negatively regulated by CaM2/3/5 and CaM7 isoforms under heat shock conditions, and that IQ6 plays an important role in CaM binding and the feedback regulation of the channel.

Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

Calcium has been implicated in the motility, assembly, disassembly, and deflagellation of the eukaryotic flagellum or cilium (exchangeable terms). Calmodulin (CaM) is known to be critical for flagellar motility; however, it is unknown whether and how CaM is involved in other flagella-related activities. We have studied CaM in Chlamydomonas, a widely used organism for ciliary studies. CaM is present in the cell body and the flagellum, with enrichment in the basal body region. Loss of CaM causes shortening of the nucleus basal body connector and impairs flagellar motility and assembly but not flagellar disassembly. Moreover, the cam mutant is defective in pH shock-induced deflagellation. The mutant deflagellates, however, upon mechanical shearing and treatment with mastoparan or detergent undergo permeabilization in the presence of calcium, indicating the cam mutant is defective in elevations of cytosolic calcium induced by pH shock, rather than by the deflagellation machinery. Indeed, the cam mutant fails to produce inositol 1,4,5-trisphosphate. Biochemical and genetic analysis showed that CaM does not directly activate PLC. Furthermore, CaM interacts with ADF1, a transient receptor channel that functions in acid-induced calcium entry. Thus, CaM is a critical regulator of flagellar activities especially those involved in modulating calcium homeostasis during acidic stress.-Wu, Q., Gao, K., Zheng, S., Zhu, X., Liang, Y., Pan, J. Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

Arabidopsis thaliana phosphoinositide-specific phospholipase C isoform 3 (AtPLC3) and AtPLC9 have an additive effect on thermotolerance.

The heat stress response is an important adaptation, enabling plants to survive challenging environmental conditions. Our previous work demonstrated that Arabidopsis thaliana Phosphoinositide-Specific Phospholipase C Isoform 9 (AtPLC9) plays an important role in thermotolerance. During prolonged heat treatment, mutants of AtPLC3 showed decreased heat resistance. We observed no obvious phenotypic differences between plc3 mutants and wild type (WT) seedlings under normal growth conditions, but after heat shock, the plc3 seedlings displayed a decline in thermotolerance compared with WT, and also showed a 40-50% decrease in survival rate and chlorophyll contents. Expression of AtPLC3 in plc3 mutants rescued the heat-sensitive phenotype; the AtPLC3-overexpressing lines also exhibited much higher heat resistance than WT and vector-only controls. The double mutants of plc3 and plc9 displayed increased sensitivity to heat stress, compared with either single mutant. In transgenic lines containing a AtPLC3:GUS promoter fusion, GUS staining showed that AtPLC3 expresses in all tissues, except anthers and young root tips. Using the Ca(2+)-sensitive fluorescent probe Fluo-3/AM and aequorin reconstitution, we showed that plc3 mutants show a reduction in the heat-induced Ca(2+) increase. The expression of HSP genes (HSP18.2, HSP25.3, HSP70-1 and HSP83) was down-regulated in plc3 mutants and up-regulated in AtPLC3-overexpressing lines after heat shock. These results indicated that AtPLC3 also plays a role in thermotolerance in Arabidopsis, and that AtPLC3 and AtPLC9 function additionally to each other.

Ubiquitin-specific proteases UBP12 and UBP13 act in circadian clock and photoperiodic flowering regulation in Arabidopsis.

Protein ubiquitination is involved in most cellular processes. In Arabidopsis (Arabidopsis thaliana), ubiquitin-mediated protein degradation regulates the stability of key components of the circadian clock feedback loops and the photoperiodic flowering pathway. Here, we identified two ubiquitin-specific proteases, UBP12 and UBP13, involved in circadian clock and photoperiodic flowering regulation. Double mutants of ubp12 and ubp13 display pleiotropic phenotypes, including early flowering and short periodicity of circadian rhythms. In ubp12 ubp13 double mutants, CONSTANS (CO) transcript rises earlier than that of wild-type plants during the day, which leads to increased expression of FLOWERING LOCUS T. This, and analysis of ubp12 co mutants, indicates that UBP12 and UBP13 regulate photoperiodic flowering through a CO-dependent pathway. In addition, UBP12 and UBP13 regulate the circadian rhythm of clock genes, including LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1, and TIMING OF CAB EXPRESSION1. Furthermore, UBP12 and UBP13 are circadian controlled. Therefore, our work reveals a role for two deubiquitinases, UBP12 and UBP13, in the control of the circadian clock and photoperiodic flowering, which extends our understanding of ubiquitin in daylength measurement in higher plants.

Phosphoinositide-specific phospholipase C9 is involved in the thermotolerance of Arabidopsis.

Intracellular calcium (Ca(2+)) increases rapidly after heat shock (HS) in the Ca(2+)/calmodulin (Ca(2+)/CaM) HS signal transduction pathway: a hypothesis proposed based on our previous findings. However, evidence for the increase in Ca(2+) after HS was obtained only through physiological and pharmacological experiments; thus, direct molecular genetic evidence is needed. The role of phosphoinositide-specific phospholipase C (PI-PLC) is poorly understood in the plant response to HS. In this work, atplc9 mutant plants displayed a serious thermosensitive phenotype compared with wild-type (WT) plants after HS. Complementation of atplc9 with AtPLC9 rescued both the basal and acquired thermotolerance phenotype of the WT plants. In addition, thermotolerance was even improved in overexpressed lines. The GUS staining of AtPLC9 promoter:GUS transgenic seedlings showed that AtPLC9 expression was ubiquitous. The fluorescence distribution of the fusion protein AtPLC9 promoter:AtPLC9:GFP revealed that the subcellular localization of AtPLC9 was restricted to the plasma membrane. The results of a PLC activity assay showed a reduction in the accumulation of inositol-1,4,5-trisphosphate (IP(3)) in atplc9 during HS and improved IP(3) generation in the overexpressed lines. Furthermore, the heat-induced increase in intracellular Ca(2+) was decreased in atplc9. Accumulation of the small HS proteins HSP18.2 and HSP25.3 was downregulated in atplc9 and upregulated in the overexpressed lines after HS. Together, these results provide molecular genetic evidence showing that AtPLC9 plays a role in thermotolerance in Arabidopsis.

A heat-activated calcium-permeable channel--Arabidopsis cyclic nucleotide-gated ion channel 6--is involved in heat shock responses.

An increased concentration of cytosolic calcium ions (Ca²âº) is an early response by plant cells to heat shock. However, the molecular mechanism underlying the heat-induced initial Ca²âº response in plants is unclear. In this study, we identified and characterized a heat-activated Ca²âº-permeable channel in the plasma membrane of Arabidopsis thaliana root protoplasts using reverse genetic analysis and the whole-cell patch-clamp technique. The results indicated that A. thaliana cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca²âº influx and facilitates expression of heat shock protein (HSP) genes and the acquisition of thermotolerance. GUS and GFP reporter assays showed that CNGC6 expression is ubiquitous in A. thaliana, and the protein is localized to the plasma membrane of cells. Furthermore, it was found that the level of cytosolic cAMP was increased by a mild heat shock, that CNGC6 was activated by cytosolic cAMP, and that exogenous cAMP promoted the expression of HSP genes. The results reveal the role of cAMP in transduction of heat shock signals in plants. The correlation of an increased level of cytosolic cAMP in a heat-shocked plant with activation of the Ca²âº channels and downstream expression of HSP genes sheds some light on how plants transduce a heat stimulus into a signal cascade that leads to a heat shock response.

The Role of Histone Modifications in Heat Signal Transduction in Plants.

Global warming and the more frequent occurrence of extremly high temperatures seriously affect crop yields. Heat stress (HS) has become a major environmental factor threatening food security worldwide. Understanding how plants sense and respond to HS is of clear interest to plant scientists and crop breeders. However, it is not trivial to elucidate the underlying signaling cascade, as specific cellular responses (ranging from detrimental to systemic effects) must be disentangled. Plants respond and adapt to high temperatures in many ways. In this review, recent progress in understanding heat signal transduction and the role of histone modifications in regulating the expression of genes involved in HS responses are discussed. The outstanding issues that are crucial for understanding the interactions between plants and HS are also discussed. The study of heat signal transduction mechanisms in plants is essential to facilitate the cultivation of heat-resistant crop varieties.

How rice adapts to high temperatures.

High-temperature stress affects crop yields worldwide. Identifying thermotolerant crop varieties and understanding the basis for this thermotolerance would have important implications for agriculture, especially in the face of climate change. Rice (Oryza sativa) varieties have evolved protective strategies to acclimate to high temperature, with different thermotolerance levels. In this review, we examine the morphological and molecular effects of heat on rice in different growth stages and plant organs, including roots, stems, leaves and flowers. We also explore the molecular and morphological differences among thermotolerant rice lines. In addition, some strategies are proposed to screen new rice varieties for thermotolerance, which will contribute to the improvement of rice for agricultural production in the future.

Boosting wheat functional genomics via an indexed EMS mutant library of KN9204.

A better understanding of wheat functional genomics can improve targeted breeding for better agronomic traits and environmental adaptation. However, the lack of gene-indexed mutants and the low transformation efficiency of wheat limit in-depth gene functional studies and genetic manipulation for breeding. In this study, we created a library for KN9204, a popular wheat variety in northern China, with a reference genome, transcriptome, and epigenome of different tissues, using ethyl methyl sulfonate (EMS) mutagenesis. This library contains a vast developmental diversity of critical tissues and transition stages. Exome capture sequencing of 2090 mutant lines using KN9204 genome-designed probes revealed that 98.79% of coding genes had mutations, and each line had an average of 1383 EMS-type SNPs. We identified new allelic variations for crucial agronomic trait-related genes such as Rht-D1, Q, TaTB1, and WFZP. We tested 100 lines with severe mutations in 80 NAC transcription factors (TFs) under drought and salinity stress and identified 13 lines with altered sensitivity. Further analysis of three lines using transcriptome and chromatin accessibility data revealed hundreds of direct NAC targets with altered transcription patterns under salt or drought stress, including SNAC1, DREB2B, CML16, and ZFP182, factors known to respond to abiotic stress. Thus, we have generated and indexed a KN9204 EMS mutant library that can facilitate functional genomics research and offer resources for genetic manipulation of wheat.

TOP1α fine-tunes TOR-PLT2 to maintain root tip homeostasis in response to sugars.

Plant development is highly dependent on energy levels. TARGET OF RAPAMYCIN (TOR) activates the proximal root meristem to promote root development in response to photosynthesis-derived sugars during photomorphogenesis in Arabidopsis thaliana. However, the mechanisms of how root tip homeostasis is maintained to ensure proper root cap structure and gravitropism are unknown. PLETHORA (PLT) transcription factors are pivotal for the root apical meristem (RAM) identity by forming gradients, but how PLT gradients are established and maintained, and their roles in COL development are not well known. We demonstrate that endogenous sucrose induces TOPOISOMERASE1α (TOP1α) expression during the skotomorphogenesis-to-photomorphogenesis transition. TOP1α fine-tunes TOR expression in the root tip columella. TOR maintains columella stem cell identity correlating with reduced quiescent centre cell division in a WUSCHEL RELATED HOMEOBOX5-independent manner. Meanwhile, TOR promotes PLT2 expression and phosphorylates and stabilizes PLT2 to maintain its gradient consistent with TOR expression pattern. PLT2 controls cell division and amyloplast formation to regulate columella development and gravitropism. This elaborate mechanism helps maintain root tip homeostasis and gravitropism in response to energy changes during root development.

The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor.

In plants, flowering time is controlled by environmental signals such as day-length and temperature, which regulate the floral pathway integrators, including FLOWERING LOCUS T (FT), by genetic and epigenetic mechanisms. Here, we identify an H3K27me3 demethylase, JUMONJI 13 (JMJ13), which regulates flowering time in Arabidopsis. Structural characterization of the JMJ13 catalytic domain in complex with its substrate peptide reveals that H3K27me3 is specifically recognized through hydrogen bonding and hydrophobic interactions. Under short-day conditions, the jmj13 mutant flowers early and has increased FT expression at high temperatures, but not at low temperatures. In contrast, jmj13 flowers early in long-day conditions regardless of temperature. Long-day condition and higher temperature induce the expression of JMJ13 and increase accumulation of JMJ13. Together, our data suggest that the H3K27me3 demethylase JMJ13 acts as a temperature- and photoperiod-dependent flowering repressor.

The role of AtPLC3 and AtPLC9 in thermotolerance in Arabidopsis.

Plants respond and adapt to temperature changes by many ways. In this article, we provide a supplement to previous work about AtPLC3. The subcellular localization showed that AtPLC3 was located in the plasma membrane and nucleus which differed from AtPLC9 localization. Furthermore, we measured the contents of IP3 before and after HS in AtPLC3 mutant, complemented and overexpressing lines. The results showed that the increase in IP3 after HS was partially dependent on AtPLC3 activity. To sum up, the similar expression patterns and phenotypes suggested that AtPLC3 and AtPLC9 may regulate the thermotolerance of Arabidopsis by the same mechanisms.

Human cytomegalovirus-encoded US28 may act as a tumor promoter in colorectal cancer.

AIM: To assess human cytomegalovirus-encoded US28 gene function in colorectal cancer (CRC) pathogenesis. METHODS: Immunohistochemical analysis was performed to determine US28 expression in 103 CRC patient samples and 98 corresponding adjacent noncancerous samples. Patient data were compared by age, sex, tumor location, histological grade, Dukes' stage, and overall mean survival time. In addition, the US28 gene was transiently transfected into the CRC LOVO cell line, and cell proliferation was assessed using a cell counting kit-8 assay. Cell cycle analysis by flow cytometry and a cell invasion transwell assay were also carried out. RESULTS: US28 levels were clearly higher in CRC tissues (38.8%) than in adjacent noncancerous samples (7.1%) (P = 0.000). Interestingly, elevated US28 amounts in CRC tissues were significantly associated with histological grade, metastasis, Dukes' stage, and overall survival (all P < 0.05); meanwhile, US28 expression was not significantly correlated with age, sex or tumor location. In addition, multivariate Cox regression data revealed US28 level as an independent CRC prognostic marker (P = 0.000). LOVO cells successfully transfected with the US28 gene exhibited higher viability, greater chemotherapy resistance, accelerated cell cycle progression, and increased invasion ability. CONCLUSION: US28 expression is predictive of poor prognosis and may promote CRC.

Molecular and genetic evidence for the key role of AtCaM3 in heat-shock signal transduction in Arabidopsis.

Heat shock (HS) is a common form of stress suffered by plants. It has been proposed that calmodulin (CaM) is involved in HS signal transduction, but direct evidence has been lacking. To investigate the potential regulatory function of CaM in the HS signal transduction pathway, T-DNA knockout mutants for AtCaM2, AtCaM3, and AtCaM4 were obtained and their thermotolerance tested. Of the three knockout mutant plants, there were no differences compared with wild-type plants under normal conditions. However, the AtCaM3 knockout mutant showed a clear reduction in thermotolerance after heat treatment at 45 degrees C for 50 min. Overexpression of AtCaM3 in either the AtCaM3 knockout or wild-type background significantly rescued or increased the thermotolerance, respectively. Results from electrophoretic mobility-shift assays, real-time quantitative reverse transcription-polymerase chain reaction, and western-blot analyses revealed that, after HS, the DNA-binding activity of HS transcription factors, mRNA transcription of HS protein genes, and accumulation of HS protein were down-regulated in the AtCaM3 knockout mutant and up-regulated in the AtCaM3-overexpressing transgenic lines. Taken together, these results suggest that endogenous AtCaM3 is a key component in the Ca2+-CaM HS signal transduction pathway.