Effects of an efficient straw decomposition system mediated by Stropharia rugosoannulata on soil properties and microbial communities in forestland.

Cultivation of Stropharia rugosoannulata with straw in forestland is effective for straw biodegradation and can prevent the waste of straw resources and environmental pollution and generate economic benefits. However, there is a lack of systematic evaluation of spent mushroom substrate (SMS) input into forestland, such as soil properties and microbial succession. In this experiment, 0 (CK), 10 (SA), 20 (SB), 30 (SC), 40 (SD), and 50 (SE) kg/m2 straw were used to cultivate S. rugosoannulata, and two soil layers (0-10 cm, 10-20 cm) of the cultivated forestland were analyzed. The results indicated that SMS significantly promoted nutrient accumulation in forestland. The bacterial alpha diversity in the SC treatment group was greater than that in the control and gradually decreased to the control level with interannual changes, while the trend of fungal alpha diversity was opposite to that of bacterial alpha diversity. Furthermore, the SC treatment group positively affected soil nitrogen metabolism-related microorganisms for two consecutive years and significantly promoted tree growth. Habitat niche breadth and null model analysis revealed that bacterial communities were more sensitive than fungal communities after SMS input. Linear mixed model (LMM) analysis revealed that SMS supplementation significantly positively affected bacteria (Gammaproteobacteria and Bacteroidota) and significantly negatively affected fungi (Coniochaetales). The constructed fungal-bacterial co-occurrence networks exhibited modularity, and the five types of bacteria were significantly correlated with soil organic matter (SOM), soil organic carbon (SOC), available potassium (AK), available phosphorus (AAP) and available nitrogen (AN) levels. The structural equation model (SEM) showed that bacterial diversity responded more to changes in soil nutrients than did fungal diversity. Overall, 30 kg/m2 of straw decomposition and 2 years of continuous cultivation were beneficial to soil health. This study provides new insights into the rational decomposition of straw and maintenance of forestland ecological balance by S. rugosoannulata.

Transcriptomic Analysis of Three Differentially Senescing Maize (Zea mays L.) Inbred Lines upon Heat Stress.

Maize, one of the world's major food crops, is facing the challenge of rising temperature. Leaf senescence is the most significant phenotypic change of maize under heat stress at the seedling stage, but the underlying molecular mechanism is still unknown. Here, we screened for three inbred lines (PH4CV, B73, and SH19B) that showed differentially senescing phenotypes under heat stress. Among them, PH4CV showed no obviously senescing phenotype under heat stress, while SH19B demonstrated a severely senescing phenotype, with B73 being between the two extremes. Subsequently, transcriptome sequencing showed that differentially expressed genes (DEGs) were generally enriched in response to heat stress, reactive oxygen species (ROS), and photosynthesis in the three inbred lines under heat treatment. Notably, ATP synthesis and oxidative phosphorylation pathway genes were only significantly enriched in SH19B. Then, the expression differences of oxidative phosphorylation pathways, antioxidant enzymes, and senescence-related genes in response to heat stress were analyzed in the three inbred lines. In addition, we demonstrated that silencing ZmbHLH51 by virus-induced gene silencing (VIGS) inhibits the heat-stress-induced senescence of maize leaves. This study helps to further elucidate the molecular mechanisms of heat-stress-induced leaf senescence at the seedling stage of maize.

Chemical induction of leaf senescence and powdery mildew resistance involves ethylene-mediated chlorophyll degradation and ROS metabolism in cucumber.

Timely initiation of leaf senescence is an integral part of plant development and, importantly, an adaptive strategy by which plants cope with various stresses, e.g. to limit the spread of pathogens. Powdery mildew is a major cucumber disease that promotes the initiation/progression of leaf senescence and reduces leaf photosynthesis, resulting in severe losses of yield and quality. However, how powdery mildew induces leaf senescence and how cucumber plants respond to enhance their resistance remain unclear. Here, with established agrochemical induction and pathogen inoculation systems, we demonstrate that both probenazole (PBZ) and powdery mildew activate ethylene (ET) biosynthesis and signal transduction, consequently promoting leaf senescence and enhancing plant resistance to powdery mildew through CsEIN3 to directly upregulate the expression of CsCCGs and CsRBOHs. Our analysis convincingly suggests that the regulation of leaf senescence and powdery mildew resistance is interconnected and mediated mainly by ET in cucumber.

The Arabidopsis IDD14 transcription factor interacts with bZIP-type ABFs/AREBs and cooperatively regulates ABA-mediated drought tolerance.

The INDETERMINATE DOMAIN (IDD) transcription factors mediate various aspects of plant growth and development. We previously reported that an Arabidopsis IDD subfamily regulates spatial auxin accumulation, and thus organ morphogenesis and gravitropic responses. However, its functions in stress responses are not well defined. Here, we use a combination of physiological, biochemical, molecular, and genetic approaches to provide evidence that the IDD14 cooperates with basic leucine zipper-type binding factors/ABA-responsive element (ABRE)-binding proteins (ABRE-binding factors (ABFs)/AREBs) in ABA-mediated drought tolerance. idd14-1D, a gain-of-function mutant of IDD14, exhibits decreased leaf water loss and improved drought tolerance, whereas inactivation of IDD14 in idd14-1 results in increased transpiration and reduced drought tolerance. Altered IDD14 expression affects ABA sensitivity and ABA-mediated stomatal closure. IDD14 can physically interact with ABF1-4 and subsequently promote their transcriptional activities. Moreover, ectopic expression and mutation of ABFs could, respectively, suppress and enhance plant sensitivity to drought stress in the idd14-1 mutant. Our results demonstrate that IDD14 forms a functional complex with ABFs and positively regulates drought-stress responses, thus revealing a previously unidentified role of IDD14 in ABA signaling and drought responses.

Transcriptome and Differentially Expressed Gene Profiles in Mycelium, Primordium and Fruiting Body Development in Stropharia rugosoannulata.

Stropharia rugosoannulata uses straw as a growth substrate during artificial cultivation and has been widely promoted in China. However, its fruiting body formation and development processes have not been elucidated. In this study, the developmental transcriptomes were analyzed at three stages: the mycelium (G-S), primordium (P-S) and fruiting body (M-F) stages. A total of 9690 differentially expressed genes (DEGs) were identified in the different developmental stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these DEGs were involved mainly in hydrolase activity, structural molecule activity and oxidoreductase activity as well as xenobiotic biodegradation and metabolism and energy metabolism pathways. We further found that the higher expression of most carbohydrate enzyme (i.e., GH, CE, CBM, AA and PL) genes in the hyphal (i.e., G-S) stage was related mainly to substrate degradation, while the upregulation of glycosyltransferase (GT) gene expression in the P-S and M-F stages may be related to cell wall synthesis. In addition, we found that CO2-sensing-related genes (i.e., CA-2, CA-3, PKA-1 and PKA-2) were upregulated in the P-S and M-F stages, heat shock protein genes (HSP60 and HSP90) were significantly downregulated in the P-S stage and upregulated in the M-F stage and the transcription factors (i.e., steA, MYB, nosA, HAP1, and GATA-4/5/6) involved in growth and development were significantly upregulated in the P-S stage. These results suggest that environmental factors (i.e., CO2 and temperature) and transcription factors may play a key role in primordium formation. In short, this study provides new insights into the study of stimulating primordia formation affecting the development of fruiting bodies of S. rugosoannulata.

Transcriptomic analysis of Stropharia rugosoannulata reveals carbohydrate metabolism and cold resistance mechanisms under low-temperature stress.

Low temperature is an important environmental factor that restricts the growth of Stropharia rugosoannulata; however, the molecular mechanisms underlying S. rugosoannulata responses to low-temperature stress are largely unknown. In this study, we performed a transcriptome analysis of a high-sensitivity strain (DQ-1) and low-sensitivity strain (DQ-3) under low-temperature stress. The liquid hyphae of S. rugosoannulata treated at 25 °C and 10 °C were analyzed by RNA-Seq, and a total of 9499 differentially expressed genes (DEGs) were identified. GO and KEGG enrichment analyses showed that these genes were enriched in "xenobiotic biodegradation and metabolism", "carbohydrate metabolism", "lipid metabolism" and "oxidoreductase activity". Further research found that carbohydrate enzyme (AA, GH, CE, and GT) genes were downregulated more significantly in DQ-1 than DQ-3 and several cellulase activities were also reduced to a greater extent. Moreover, the CAT1, CAT2, GR, and POD genes and more heat shock protein genes (HSP20, HSP78 and sHSP) were upregulated in the two strains after low-temperature stress, and the GPX gene and more heat shock protein genes were upregulated in DQ-3. In addition, the enzyme activity and qRT-PCR results showed trends similar to those of the RNA-Seq results. This result indicates that low-temperature stress reduces the expression of different AA, GH, CE, and GT enzyme genes and reduces the secretion of cellulase, thereby reducing the carbohydrate metabolism process and mycelial growth of S. rugosoannulata. Moreover, the expression levels of different types of antioxidant enzymes and heat shock proteins are also crucial for S. rugosoannulata to resist low-temperature stress. In short, this study will provide a basis for further research on important signaling pathways, gene functions and variety breeding of S. rugosoannulata related to low-temperature stress.

The NPR1-WRKY46-WRKY6 signaling cascade mediates probenazole/salicylic acid-elicited leaf senescence in Arabidopsis thaliana.

Endogenous salicylic acid (SA) regulates leaf senescence, but the underlying mechanism remains largely unexplored. The exogenous application of SA to living plants is not efficient for inducing leaf senescence. By taking advantage of probenazole (PBZ)-induced biosynthesis of endogenous SA, we previously established a chemical inducible leaf senescence system that depends on SA biosynthesis and its core signaling receptor NPR1 in Arabidopsis thaliana. Here, using this system, we identified WRKY46 and WRKY6 as key components of the transcriptional machinery downstream of NPR1 signaling. Upon PBZ treatment, the wrky46 mutant exhibited significantly delayed leaf senescence. We demonstrate that NPR1 is essential for PBZ/SA-induced WRKY46 activation, whereas WRKY46 in turn enhances NPR1 expression. WRKY46 interacts with NPR1 in the nucleus, binding to the W-box of the WRKY6 promoter to induce its expression in response to SA signaling. Dysfunction of WRKY6 abolished PBZ-induced leaf senescence, while overexpression of WRKY6 was sufficient to accelerate leaf senescence even under normal growth conditions, suggesting that WRKY6 may serve as an integration node of multiple leaf senescence signaling pathways. Taken together, these findings reveal that the NPR1-WRKY46-WRKY6 signaling cascade plays a critical role in PBZ/SA-mediated leaf senescence in Arabidopsis.

Correction to: MKK4/MKK5­MPK1/MPK2 cascade mediates SA­activated leaf senescence via phosphorylation of NPR1 in Arabidopsis.

Due to an unfortunate turn of events, the second co-corresponding author, Dr. Benke Kuai, was omitted from the original publication. The corrected authors' list and author contribution statement are published here and should be treated as definitive.

Hyponastic Leaves 1 protects pri-miRNAs from nuclear exosome attack.

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.

The transcription factor ZmNAC126 accelerates leaf senescence downstream of the ethylene signalling pathway in maize.

Leaf senescence is an integral part of plant development, during which, nutrients are remobilized from senescent leaves to fast-growing organs. The initiation and progression dynamics of leaf senescence is therefore vital not only to the maximal accumulation of assimilates but also to the efficient remobilization of nutrients. Senescence is a finely tuned process that involves the action of a large number of transcription factors (TFs). The NAC TFs play critical roles in regulating leaf senescence in Arabidopsis, wheat, rice and tomato. Here, we identified a NAC TF, ZmNAC126 that is responsive to leaf senescence in maize. Ectopic overexpression of ZmNAC126 in Arabidopsis and maize enhanced chlorophyll degradation and promoted leaf senescence. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that ZmNAC126 could directly bind to the promoters of major chlorophyll catabolic genes in maize. Dual-luciferase assay in maize protoplasts indicated that ZmNAC126 positively regulates these chlorophyll catabolic genes in maize. Moreover, ZmNAC126 could be induced by ethylene, and ZmEIN3, a major TF of ethylene signalling, could bind to its promoter to transactivate its expression. Taken together, ZmNAC126 may play a pivotal role in regulating natural and ethylene-triggered leaf senescence in maize.

Overexpression of TCP8 delays Arabidopsis flowering through a FLOWERING LOCUS C-dependent pathway.

BACKGROUND: Flowering is a key process in the life cycle of plants. The transition from vegetative to reproductive growth is thus under sophisticated regulation by endogenous and environmental signals. The plant-specific Teosinte Branched 1/Cycloidea/Proliferating Cell Factors (TCP) family transcription factors are involved in many biological processes, but their roles in regulating flowering have not been totally elucidated. RESULTS: We explored the role of Arabidopsis TCP8 in plant development and, especially, in flowering control. Overexpression of TCP8 significantly delayed flowering under both long-day and short-day conditions and dominant repression by TCP8 led to various growth defects. The upregulation of TCP8 led to more accumulated mRNA level of FLOWERING LOCUS C (FLC), a central floral repressor of Arabidopsis. TCP8 functions in an FLC-dependent manner, as TCP8 overexpression in the flc-6 loss-of-function mutant failed to delay flowering. The vernalization treatment could reverse the late flowering phenotype caused by TCP8 overexpression. CONCLUSIONS: Our results provide evidence for a role of TCP8 in flowering control and add to our knowledge of the molecular basis of TCP8 function.

The C-terminal cysteine-rich motif of NYE1/SGR1 is indispensable for its function in chlorophyll degradation in Arabidopsis.

KEY MESSAGE: The C-terminal cysteine-rich motif of NYE1/SGR1 affects chlorophyll degradation likely by mediating its self-interaction and conformational change, and somehow altering its Mg-dechelating activity in response to the changing redox potential. During green organ senescence in plants, the most prominent phenomenon is the degreening caused by net chlorophyll (Chl) loss. NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) was recently reported to be able to dechelates magnesium (Mg) from Chl a to initiate its degradation, but little is known about the domain/motif basis of its functionality. In this study, we carried out a protein truncation assay and identified a conserved cysteine-rich motif (CRM, P-X3-C-X3-C-X-C2-F-P-X5-P) at its C terminus, which is essential for its function. Genetic analysis showed that all four cysteines in the CRM were irreplaceable, and enzymatic assays demonstrated that the mutation of each of the four cysteines affected its Mg-dechelating activity. The CRM plays a critical role in the conformational change and self-interaction of NYE1 via the formation of inter- and intra-molecular disulfide bonds. Our results may provide insight into how NYE1 responds to rapid redox changes during leaf senescence and in response to various environmental stresses.

The H3K27me3 demethylase REF6 promotes leaf senescence through directly activating major senescence regulatory and functional genes in Arabidopsis.

The roles of histone demethylation in the regulation of plant flowering, disease resistance, rhythmical response, and seed germination have been elucidated recently; however, how histone demethylation affects leaf senescence remains largely unclear. In this study, we exploited yeast one-hybrid (Y1H) to screen for the upstream regulators of NONYELLOWING1 (NYE1), and identified RELATIVE OF EARLY FLOWERING6 (REF6), a histone H3 lysine 27 tri-methylation (H3K27me3) demethylase, as a putative binding protein of NYE1 promoter. By in vivo and in vitro analyses, we demonstrated that REF6 directly binds to the motif CTCGYTY in NYE1/2 promoters through its zinc finger domain and positively regulates their expression. Loss-of-function of REF6 delayed chlorophyll (Chl) degradation, whereas overexpression of REF6 accelerated Chl degradation. Subsequently, we revealed that REF6 positively regulates the general senescence process by directly up-regulating ETHYLENE INSENSITIVE 2 (EIN2), ORESARA1 (ORE1), NAC-LIKE, ACTIVATED BY AP3/PI (NAP), PYRUVATE ORTHOPHOSPHATE DIKINASE (PPDK), PHYTOALEXIN DEFICIENT 4 (PAD4), LIPOXYGENASE 1 (LOX1), NAC DOMAIN CONTAINING PROTEIN 3 (AtNAC3), and NAC TRANSCRIPTION FACTOR-LIKE 9 (NTL9), the key regulatory and functional genes predominantly involved in the regulation of developmental leaf senescence. Importantly, loss-of-function of REF6 increased H3K27me3 levels at all the target Senescence associated genes (SAGs). We therefore conclusively demonstrate that H3K27me3 methylation represents an epigenetic mechanism prohibiting the premature transcriptional activation of key developmentally up-regulated senescence regulatory as well as functional genes in Arabidopsis.

REF6 promotes lateral root formation through de-repression of PIN1/3/7 genes.

The H3K27 methyltransferase CLF inhibits lateral root (LR) formation through depositing the repressive H3K27me3 mark to the chromatin of PIN1, a key polar auxin transporter gene. Here, we show that the H3K27me3 demethylase REF6 promotes lateral root primordium initiation and LR emergence. REF6 directly binds to the chromatin of PIN1/3/7. Dysfunction in REF6 results in increased levels of H3K27me3 on PIN1/3/7 and suppressed expression of PIN genes. Genetic analysis of the clf ref6 double mutant revealed an antagonistic action between CLF and REF6, in terms of LR formation. Our findings indicate that H3K27 methylation and demethylation activities are likely coordinated to ensure proper LR organogenesis.

CIRCADIAN CLOCK-ASSOCIATED 1 Inhibits Leaf Senescence in Arabidopsis.

Leaf senescence is an integral part of plant development, and the timing and progressing rate of senescence could substantially affect the yield and quality of crops. It has been known that a circadian rhythm synchronized with external environmental cues is critical for the optimal coordination of various physiological and metabolic processes. However, the reciprocal interactions between the circadian clock and leaf senescence in plants remain unknown. Here, through measuring the physiological and molecular senescence related markers of several circadian components mutants, we found that CIRCADIAN CLOCK-ASSOCIATED 1 inhibits leaf senescence. Further molecular and genetic studies revealed that CCA1 directly activates GLK2 and suppresses ORE1 expression to counteract leaf senescence. As plants age, the expression and periodic amplitude of CCA1 declines and thus weakens the inhibition of senescence. Our findings reveal an age-dependent circadian clock component of the process of leaf senescence.

The biochemistry and molecular biology of chlorophyll breakdown.

Chlorophyll breakdown is one of the most obvious signs of leaf senescence and fruit ripening. The resulting yellowing of leaves can be observed every autumn, and the color change of fruits indicates their ripening state. During these processes, chlorophyll is broken down in a multistep pathway, now termed the 'PAO/phyllobilin' pathway, acknowledging the core enzymatic breakdown step catalysed by pheophorbide a oxygenase, which determines the basic linear tetrapyrrole structure of the products of breakdown that are now called 'phyllobilins'. This review provides an update on the PAO/phyllobilin pathway, and focuses on recent biochemical and molecular progress in understanding phyllobilin-modifying reactions as the basis for phyllobilin diversity, on the evolutionary diversity of the pathway, and on the transcriptional regulation of the pathway genes.

Phytohormone and Light Regulation of Chlorophyll Degradation.

Degreening, due to the net loss of chlorophyll (Chl), is the most prominent symptom during the processes of leaf senescence, fruit ripening, and seed maturation. Over the last decade or so, extensive identifications of Chl catabolic genes (CCGs) have led to the revelation of the biochemical pathway of Chl degradation. As such, exploration of the regulatory mechanism of the degreening process is greatly facilitated. During the past few years, substantial progress has been made in elucidating the regulation of Chl degradation, particularly via the mediation of major phytohormones' signaling. Intriguingly, ethylene and abscisic acid's signaling have been demonstrated to interweave with light signaling in mediating the regulation of Chl degradation. In this review, we briefly summarize this progress, with an effort on providing a framework for further investigation of multifaceted and hierarchical regulations of Chl degradation.

NYEs/SGRs-mediated chlorophyll degradation is critical for detoxification during seed maturation in Arabidopsis.

In the seed industry, chlorophyll (Chl) fluorescence is often used as a major non-invasive reporter of seed maturation and quality. Breakdown of Chl is a proactive process during the late stage of seed maturation, as well as during leaf senescence and fruit ripening. However, the biological significance of this process is still unclear. NYE1 and NYE2 are Mg-dechelatases, catalyzing the first rate-limiting step of Chl a degradation. Loss-of-function of both NYE1 and NYE2 not only results in a nearly complete retention of Chl during leaf senescence, but also produces green seeds in Arabidopsis. In this study, we showed that Chl retention in the nye1 nye2 double-mutant caused severe photo-damage to maturing seeds. Upon prolonged light exposure, green seeds of nye1 nye2 gradually bleached out and eventually lost their germination capacity. This organ-specific photosensitive phenotype is likely due to an over-accumulation of free Chl, which possesses photosensitizing properties and causes a burst of reactive oxygen species upon light exposure. As expected, a similar, albeit much milder, photosensitive phenotype was observed in the seeds of d1 d2, a green-seed mutant defective in NYE/SGR orthologous genes in soybean. Taken together, our data suggest that efficient NYEs-mediated Chl degradation is critical for detoxification during seed maturation.

Suppressor of Overexpression of CO 1 Negatively Regulates Dark-Induced Leaf Degreening and Senescence by Directly Repressing Pheophytinase and Other Senescence-Associated Genes in Arabidopsis.

Although the biochemical pathway of chlorophyll (Chl) degradation has been largely elucidated, how Chl is rapidly yet coordinately degraded during leaf senescence remains elusive. Pheophytinase (PPH) is the enzyme for catalyzing the removal of the phytol group from pheophytin a, and PPH expression is significantly induced during leaf senescence. To elucidate the transcriptional regulation of PPH, we used a yeast (Saccharomyces cerevisiae) one-hybrid system to screen for its trans-regulators. SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), a key flowering pathway integrator, was initially identified as one of the putative trans-regulators of PPH After dark treatment, leaves of an SOC1 knockdown mutant (soc1-6) showed an accelerated yellowing phenotype, whereas those of SOC1-overexpressing lines exhibited a partial stay-green phenotype. SOC1 and PPH expression showed a negative correlation during leaf senescence. Substantially, SOC1 protein could bind specifically to the CArG box of the PPH promoter in vitro and in vivo, and overexpression of SOC1 significantly inhibited the transcriptional activity of the PPH promoter in Arabidopsis (Arabidopsis thaliana) protoplasts. Importantly, soc1-6 pph-1 (a PPH knockout mutant) double mutant displayed a stay-green phenotype similar to that of pph-1 during dark treatment. These results demonstrated that SOC1 inhibits Chl degradation via negatively regulating PPH expression. In addition, measurement of the Chl content and the maximum photochemical efficiency of photosystem II of soc1-6 and SOC1-OE leaves after dark treatment suggested that SOC1 also negatively regulates the general senescence process. Seven SENESCENCE-ASSOCIATED GENES (SAGs) were thereafter identified as its potential target genes, and NONYELLOWING1 and SAG113 were experimentally confirmed. Together, we reveal that SOC1 represses dark-induced leaf Chl degradation and senescence in general in Arabidopsis.