An RRM domain protein SOE suppresses transgene silencing in rice.

Gene expression is regulated at multiple levels, including RNA processing and DNA methylation/demethylation. How these regulations are controlled remains unclear. Here, through analysis of a suppressor for the OsEIN2 over-expressor, we identified an RNA recognition motif protein SUPPRESSOR OF EIN2 (SOE). SOE is localized in nuclear speckles and interacts with several components of the spliceosome. We find SOE associates with hundreds of targets and directly binds to a DNA glycosylase gene DNG701 pre-mRNA for efficient splicing and stabilization, allowing for subsequent DNG701-mediated DNA demethylation of the transgene promoter for proper gene expression. The V81M substitution in the suppressor mutant protein mSOE impaired its protein stability and binding activity to DNG701 pre-mRNA, leading to transgene silencing. SOE mutation enhances grain size and yield. Haplotype analysis in c. 3000 rice accessions reveals that the haplotype 1 (Hap 1) promoter is associated with high 1000-grain weight, and most of the japonica accessions, but not indica ones, have the Hap 1 elite allele. Our study discovers a novel mechanism for the regulation of gene expression and provides an elite allele for the promotion of yield potentials in rice.

Regulation of seed traits in soybean.

Soybean (Glycine max) is an essential economic crop that provides vegetative oil and protein for humans, worldwide. Increasing soybean yield as well as improving seed quality is of great importance. Seed weight/size, oil and protein content are the three major traits determining seed quality, and seed weight also influences soybean yield. In recent years, the availability of soybean omics data and the development of related techniques have paved the way for better research on soybean functional genomics, providing a comprehensive understanding of gene functions. This review summarizes the regulatory genes that influence seed size/weight, oil content and protein content in soybean. We also provided a general overview of the pleiotropic effect for the genes in controlling seed traits and environmental stresses. Ultimately, it is expected that this review will be beneficial in breeding improved traits in soybean.

Global analysis of seed transcriptomes reveals a novel PLATZ regulator for seed size and weight control in soybean.

Seed size and weight are important factors that influence soybean yield. Combining the weighted gene co-expression network analysis (WGCNA) of 45 soybean accessions and gene dynamic changes in seeds at seven developmental stages, we identified candidate genes that may control the seed size/weight. Among these, a PLATZ-type regulator overlapping with 10 seed weight QTLs was further investigated. This zinc-finger transcriptional regulator, named as GmPLATZ, is required for the promotion of seed size and weight in soybean. The GmPLATZ may exert its functions through direct binding to the promoters and activation of the expression of cyclin genes and GmGA20OX for cell proliferation. Overexpression of the GmGA20OX enhanced seed size/weight in soybean. We further found that the GmPLATZ binds to a 32-bp sequence containing a core palindromic element AATGCGCATT. Spacing of the flanking sequences beyond the core element facilitated GmPLATZ binding. An elite haplotype Hap3 was also identified to have higher promoter activity and correlated with higher gene expression and higher seed weight. Orthologues of the GmPLATZ from rice and Arabidopsis play similar roles in seeds. Our study reveals a novel module of GmPLATZ-GmGA20OX/cyclins in regulating seed size and weight and provides valuable targets for breeding of crops with desirable agronomic traits.

A translational regulator MHZ9 modulates ethylene signaling in rice.

Ethylene plays essential roles in rice growth, development and stress adaptation. Translational control of ethylene signaling remains unclear in rice. Here, through analysis of an ethylene-response mutant mhz9, we identified a glycine-tyrosine-phenylalanine (GYF) domain protein MHZ9, which positively regulates ethylene signaling at translational level in rice. MHZ9 is localized in RNA processing bodies. The C-terminal domain of MHZ9 interacts with OsEIN2, a central regulator of rice ethylene signaling, and the N-terminal domain directly binds to the OsEBF1/2 mRNAs for translational inhibition, allowing accumulation of transcription factor OsEIL1 to activate the downstream signaling. RNA-IP seq and CLIP-seq analyses reveal that MHZ9 associates with hundreds of RNAs. Ribo-seq analysis indicates that MHZ9 is required for the regulation of ~ 90% of genes translationally affected by ethylene. Our study identifies a translational regulator MHZ9, which mediates translational regulation of genes in response to ethylene, facilitating stress adaptation and trait improvement in rice.

GmJAZ3 interacts with GmRR18a and GmMYC2a to regulate seed traits in soybean.

Seed weight is usually associated with seed size and is one of the important agronomic traits that determine yield. Understanding of seed weight control is limited, especially in soybean plants. Here we show that Glycine max JASMONATE-ZIM DOMAIN 3 (GmJAZ3), a gene identified through gene co-expression network analysis, regulates seed-related traits in soybean. Overexpression of GmJAZ3 promotes seed size/weight and other organ sizes in stable transgenic soybean plants likely by increasing cell proliferation. GmJAZ3 interacted with both G. max RESPONSE REGULATOR 18a (GmRR18a) and GmMYC2a to inhibit their transcriptional activation of cytokinin oxidase gene G. max CYTOKININ OXIDASE 3-4 (GmCKX3-4), which usually affects seed traits. Meanwhile, the GmRR18a binds to the promoter of GmMYC2a and activates GmMYC2a gene expression. In GmJAZ3-overexpressing soybean seeds, the protein contents were increased while the fatty acid contents were reduced compared to those in the control seeds, indicating that the GmJAZ3 affects seed size/weight and compositions. Natural variation in JAZ3 promoter region was further analyzed and Hap3 promoter correlates with higher promoter activity, higher gene expression and higher seed weight. The Hap3 promoter may be selected and fixed during soybean domestication. JAZ3 orthologs from other plants/crops may also control seed size and weight. Taken together, our study reveals a novel molecular module GmJAZ3-GmRR18a/GmMYC2a-GmCKXs for seed size and weight control, providing promising targets during soybean molecular breeding for better seed traits.

Zinc-finger protein GmZF351 improves both salt and drought stress tolerance in soybean.

Abiotic stress is one of the most important factors reducing soybean yield. It is essential to identify regulatory factors contributing to stress responses. A previous study found that the tandem CCCH zinc-finger protein GmZF351 is an oil level regulator. In this study, we discovered that the GmZF351 gene is induced by stress and that the overexpression of GmZF351 confers stress tolerance to transgenic soybean. GmZF351 directly regulates the expression of GmCIPK9 and GmSnRK, leading to stomata closing, by binding to their promoter regions, which carry two CT(G/C)(T/A)AA elements. Stress induction of GmZF351 is mediated through reduction in the H3K27me3 level at the GmZF351 locus. Two JMJ30-demethylase-like genes, GmJMJ30-1 and GmJMJ30-2, are involved in this demethylation process. Overexpression of GmJMJ30-1/2 in transgenic hairy roots enhances GmZF351 expression mediated by histone demethylation and confers stress tolerance to soybean. Yield-related agronomic traits were evaluated in stable GmZF351-transgenic plants under mild drought stress conditions. Our study reveals a new mode of GmJMJ30-GmZF351 action in stress tolerance, in addition to that of GmZF351 in oil accumulation. Manipulation of the components in this pathway is expected to improve soybean traits and adaptation under unfavorable environments.

Melatonin regulates gene expressions through activating auxin synthesis and signaling pathways.

Background: Both melatonin and indole-3-acetic acid (IAA) are derived from tryptophan. And the most interesting and unsolved puzzle in melatonin research is that what is the relationship between melatonin and auxin? Methods: In this study, we performed transcriptome analysis with a time series method to disclose the connection of the two metabolites in soybean. Results: Our results reveal that melatonin and IAA treatments cause substantial overlaps in gene expression changes. Common genes of melatonin and IAA treatments could be sorted into clusters with very similar expression tendency. A KEGG assay showed that exogenous applied melatonin enriched differentially expressed genes in auxin biosynthesis and signaling pathways. For details, melatonin up-regulates several YUCCA genes which participate in auxin biosynthesis; melatonin also enhances expression levels of auxin receptor coding genes, such as TIR1, AFB3 and AFB5; dozens of genes involved in auxin transport, such as AUXI and PIN, are regulated by melatonin similarly as by auxin; auxin-responsive genes, such as IAA, ARF, GH3 and SAUR-like genes, intensively respond to melatonin as well as to auxin. A DR5 promoter mediated GUS staining assay showed that low concentration of melatonin could induce auxin biosynthesis in a dosage manner, whereas high concentration of melatonin would eliminate such effect. At last, gene ontology (GO) analysis suggests that melatonin treatment has similar characteristics as auxin treatment in many processes. However, the two molecules still keep their own features respectively. For example, melatonin takes part in stress responses, while IAA treatment enriches the GO terms that related to cell growth. Conclusion: Taken together, exogenous applied melatonin, if not exceeds the appropriate concentration, could promote auxin responses range from biosynthesis to signaling transduction. Thus, our research is a key part to explain the auxin-like roles of melatonin in regulating plant growth.

Rice EIL1 interacts with OsIAAs to regulate auxin biosynthesis mediated by the tryptophan aminotransferase MHZ10/OsTAR2 during root ethylene responses.

Ethylene plays essential roles in adaptive growth of rice (Oryza sativa). Understanding of the crosstalk between ethylene and auxin (Aux) is limited in rice. Here, from an analysis of the root-specific ethylene-insensitive rice mutant mao hu zi 10 (mhz10), we identified the tryptophan aminotransferase (TAR) MHZ10/OsTAR2, which catalyzes the key step in indole-3-pyruvic acid-dependent Aux biosynthesis. Genetically, OsTAR2 acts downstream of ethylene signaling in root ethylene responses. ETHYLENE INSENSITIVE3 like1 (OsEIL1) directly activated OsTAR2 expression. Surprisingly, ethylene induction of OsTAR2 expression still required the Aux pathway. We also show that Os indole-3-acetic acid (IAA)1/9 and OsIAA21/31 physically interact with OsEIL1 and show promotive and repressive effects on OsEIL1-activated OsTAR2 promoter activity, respectively. These effects likely depend on their EAR motif-mediated histone acetylation/deacetylation modification. The special promoting activity of OsIAA1/9 on OsEIL1 may require both the EAR motifs and the flanking sequences for recruitment of histone acetyltransferase. The repressors OsIAA21/31 exhibit earlier degradation upon ethylene treatment than the activators OsIAA1/9 in a TIR1/AFB-dependent manner, allowing OsEIL1 activation by activators OsIAA1/9 for OsTAR2 expression and signal amplification. This study reveals a positive feedback regulation of ethylene signaling by Aux biosynthesis and highlights the crosstalk between ethylene and Aux pathways at a previously underappreciated level for root growth regulation in rice.

Nuclear factor Y subunit GmNFYA competes with GmHDA13 for interaction with GmFVE to positively regulate salt tolerance in soybean.

Soybean is an important crop worldwide, but its production is severely affected by salt stress. Understanding the regulatory mechanism of salt response is crucial for improving the salt tolerance of soybean. Here, we reveal a role for nuclear factor Y subunit GmNFYA in salt tolerance of soybean likely through the regulation of histone acetylation. GmNFYA is induced by salt stress. Overexpression of GmNFYA significantly enhances salt tolerance in stable transgenic soybean plants by inducing salt-responsive genes. Analysis in soybean plants with transgenic hairy roots also supports the conclusion. GmNFYA interacts with GmFVE, which functions with putative histone deacetylase GmHDA13 in a complex for transcriptional repression possibly by reducing H3K9 acetylation at target loci. Under salt stress, GmNFYA likely accumulates and competes with GmHDA13 for interaction with GmFVE, leading to the derepression and maintenance of histone acetylation for activation of salt-responsive genes and finally conferring salt tolerance in soybean plants. In addition, a haplotype I GmNFYA promoter is identified with the highest self-activated promoter activity and may be selected during future breeding for salt-tolerant cultivars. Our study uncovers the epigenetic regulatory mechanism of GmNFYA in salt-stress response, and all the factors/elements identified may be potential targets for genetic manipulation of salt tolerance in soybean and other crops.

A transcriptional regulatory module controls lipid accumulation in soybean.

Soybean (Glycine max) is one of the most important oilseed crops. However, the regulatory mechanism that governs the process of oil accumulation in soybean remains poorly understood. In this study, GmZF392, a tandem CCCH zinc finger (TZF) protein which was identified in our previous RNA-seq analysis of seed-preferred transcription factors, was found to function as a positive regulator of lipid production. GmZF392 promotes seed oil accumulation in both transgenic Arabidopsis and stable transgenic soybean plants by binding to a bipartite cis-element, containing TG- and TA-rich sequences, in promoter regions, activating the expression of genes in the lipid biosynthesis pathway. GmZF392 physically interacts with GmZF351, our previously identified transcriptional regulator of lipid biosynthesis, to synergistically promote downstream gene expression. Both GmZF392 and GmZF351 are further upregulated by GmNFYA, another transcription factor involved in lipid biosynthesis, directly (in the former case) and indirectly (in the latter case). Promoter sequence diversity analysis showed that the GmZF392 promoter may have been selected at the origin of the Glycine genus and further mildly selected during domestication from wild soybeans to cultivated soybeans. Our study reveals a regulatory module containing three transcription factors in the lipid biosynthesis pathway, and manipulation of the module may improve oil production in soybean and other oilseed crops.

The GDSL Lipase MHZ11 Modulates Ethylene Signaling in Rice Roots.

Ethylene plays important roles in plant growth and development, but the regulation of ethylene signaling is largely unclear, especially in crops such as rice (Oryza sativa). Here, by analysis of the ethylene-insensitive mutant mao huzi 11 (mhz11), we identified the GDSL lipase MHZ11, which modulates ethylene signaling in rice roots. MHZ11 localized to the endoplasmic reticulum membrane and has acyl-hydrolyzing activity. This activity affects the homeostasis of sterols in rice roots and is required for root ethylene response. MHZ11 overexpression caused constitutive ethylene response in roots. Genetically, MHZ11 acts with the ethylene receptor ETHYLENE RESPONSE SENSOR2 (OsERS2) upstream of CONSTITUTIVE TRIPLE RESPONSE2 (OsCTR2) and ETHYLENE INSENSITIVE2 (OsEIN2). The mhz11 mutant maintains more OsCTR2 in the phosphorylated form whereas MHZ11 overexpression promotes ethylene-mediated inhibition of OsCTR2 phosphorylation. MHZ11 colocalized with the ethylene receptor OsERS2, and its effect on OsCTR2 phosphorylation requires ethylene perception and initiation of ethylene signaling. The mhz11 mutant overaccumulated sterols and blocking sterol biosynthesis partially rescued the mhz11 ethylene response, likely by reducing receptor-OsCTR2 interaction and OsCTR2 phosphorylation. We propose that MHZ11 reduces sterol levels to impair receptor-OsCTR2 interactions and OsCTR2 phosphorylation for triggering ethylene signaling. Our study reveals a mechanism by which MHZ11 participates in ethylene signaling for regulation of root growth in rice.

Histidine kinase MHZ1/OsHK1 interacts with ethylene receptors to regulate root growth in rice.

Ethylene plays essential roles during adaptive responses to water-saturating environments in rice, but knowledge of its signaling mechanism remains limited. Here, through an analysis of a rice ethylene-response mutant mhz1, we show that MHZ1 positively modulates root ethylene responses. MHZ1 encodes the rice histidine kinase OsHK1. MHZ1/OsHK1 is autophosphorylated at a conserved histidine residue and can transfer the phosphoryl signal to the response regulator OsRR21 via the phosphotransfer proteins OsAHP1/2. This phosphorelay pathway is required for root ethylene responses. Ethylene receptor OsERS2, via its GAF domain, physically interacts with MHZ1/OsHK1 and inhibits its kinase activity. Genetic analyses suggest that MHZ1/OsHK1 acts at the level of ethylene perception and works together with the OsEIN2-mediated pathway to regulate root growth. Our results suggest that MHZ1/OsHK1 mediates the ethylene response partially independently of OsEIN2, and is directly inhibited by ethylene receptors, thus revealing mechanistic details of ethylene signaling for root growth regulation.

A class B heat shock factor selected for during soybean domestication contributes to salt tolerance by promoting flavonoid biosynthesis.

Soybean (Glycine max) production is severely affected in unfavorable environments. Identification of the regulatory factors conferring stress tolerance would facilitate soybean breeding. In this study, through coexpression network analysis of salt-tolerant wild soybeans, together with molecular and genetic approaches, we revealed a previously unidentified function of a class B heat shock factor, HSFB2b, in soybean salt stress response. We showed that HSFB2b improves salt tolerance through the promotion of flavonoid accumulation by activating one subset of flavonoid biosynthesis-related genes and by inhibiting the repressor gene GmNAC2 to release another subset of genes in the flavonoid biosynthesis pathway. Moreover, four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II from salt-tolerant wild soybean Y20, with high promoter activity under salt stress, is probably selected for during domestication. Another promoter haplotype, III, from salt-tolerant wild soybean Y55, had the highest promoter activity under salt stress, had a low distribution frequency and may be subjected to the next wave of selection. Together, our results revealed the mechanism of HSFB2b in soybean salt stress tolerance. Its promoter variations were identified, and the haplotype with high activity may be adopted for breeding better soybean cultivars that are adapted to stress conditions.

GmWRKY54 improves drought tolerance through activating genes in abscisic acid and Ca2+ signaling pathways in transgenic soybean.

WRKY transcription factors play important roles in response to various abiotic stresses. Previous study have proved that soybean GmWRKY54 can improve stress tolerance in transgenic Arabidopsis. Here, we generated soybean transgenic plants and further investigated roles and biological mechanisms of GmWRKY54 in response to drought stress. We demonstrated that expression of GmWRKY54, driven by either a constitutive promoter (pCm) or a drought-induced promoter (RD29a), confers drought tolerance. GmWRKY54 is a transcriptional activator and affects a large number of stress-related genes as revealed by RNA sequencing. Gene ontology (GO) enrichment and co-expression network analysis, together with measurement of physiological parameters, supported the idea that GmWRKY54 enhances stomatal closure to reduce water loss, and therefore confers drought tolerance in soybean. GmWRKY54 directly binds to the promoter regions of genes including PYL8, SRK2A, CIPK11 and CPK3 and activates them. Therefore GmWRKY54 achieves its function through abscisic acid (ABA) and Ca2+ signaling pathways. It is valuable that GmWRKY54 activates an ABA receptor and an SnRK2 kinase in the upstream position, unlike other WRKY proteins that regulate downstream genes in the ABA pathway. Our study revealed the role of GmWRKY54 in drought tolerance and further manipulation of this gene should improve growth and production in soybean and other legumes/crops under unfavorable conditions.

Membrane protein MHZ3 stabilizes OsEIN2 in rice by interacting with its Nramp-like domain.

The phytohormone ethylene regulates many aspects of plant growth and development. EIN2 is the central regulator of ethylene signaling, and its turnover is crucial for triggering ethylene responses. Here, we identified a stabilizer of OsEIN2 through analysis of the rice ethylene-response mutant mhz3. Loss-of-function mutations lead to ethylene insensitivity in etiolated rice seedlings. MHZ3 encodes a previously uncharacterized membrane protein localized to the endoplasmic reticulum. Ethylene induces MHZ3 gene and protein expression. Genetically, MHZ3 acts at the OsEIN2 level in the signaling pathway. MHZ3 physically interacts with OsEIN2, and both the N- and C-termini of MHZ3 specifically associate with the OsEIN2 Nramp-like domain. Loss of mhz3 function reduces OsEIN2 abundance and attenuates ethylene-induced OsEIN2 accumulation, whereas MHZ3 overexpression elevates the abundance of both wild-type and mutated OsEIN2 proteins, suggesting that MHZ3 is required for proper accumulation of OsEIN2 protein. The association of MHZ3 with the Nramp-like domain is crucial for OsEIN2 accumulation, demonstrating the significance of the OsEIN2 transmembrane domains in ethylene signaling. Moreover, MHZ3 negatively modulates OsEIN2 ubiquitination, protecting OsEIN2 from proteasome-mediated degradation. Together, these results suggest that ethylene-induced MHZ3 stabilizes OsEIN2 likely by binding to its Nramp-like domain and impeding protein ubiquitination to facilitate ethylene signal transduction. Our findings provide insight into the mechanisms of ethylene signaling.

An Alfin-like gene from Atriplex hortensis enhances salt and drought tolerance and abscisic acid response in transgenic Arabidopsis.

Alfin-like (AL) is a small plant-specific gene family with prominent roles in root growth and abiotic stress response. Here, we aimed to identify novel stress tolerance AL genes from the stress-tolerant species Atriplex hortensis. Totally, we isolated four AhAL genes, all encoding nuclear-localized proteins with cis-element-binding and transrepression activities. Constitutive expression of AhAL1 in Arabidopsis facilitated plants to survive under saline condition, while expressing anyone of the other three AhAL genes led to salt-hypersensitive response, indicating functional divergence of AhAL family. AhAL1 also conferred enhanced drought tolerance, as judged from enhanced survival, improved growth, decreased malonaldehyde (MDA) content and reduced water loss in AhAL1-expressing plants compared to WT. In addition, abscisic acid (ABA)-mediated stomatal closure and inhibition of seed germination and primary root elongation were enhanced in AhAL1-transgenic plants. Further analysis demonstrated that AhAL1 could bind to promoter regions of GRF7, DREB1C and several group-A PP2C genes and repress their expression. Correspondingly, the expression levels of positive stress regulator genes DREB1A, DREB2A and three ABFs were all increased in AhAL1-expressing plants. Based on these results, AhAL1 was identified as a novel candidate gene for improving abiotic stress tolerance of crop plants.

A Histone Code Reader and a Transcriptional Activator Interact to Regulate Genes for Salt Tolerance.

Plant homeodomain (PHD) finger proteins are involved in various developmental processes and stress responses. They recognize and bind to epigenetically modified histone H3 tail and function as histone code readers. Here we report that GmPHD6 reads low methylated histone H3K4me0/1/2 but not H3K4me3 with its N-terminal domain instead of the PHD finger. GmPHD6 does not possess transcriptional regulatory ability but has DNA-binding ability. Through the PHD finger, GmPHD6 interacts with its coactivator, LHP1-1/2, to form a transcriptional activation complex. Using a transgenic hairy root system, we demonstrate that overexpression of GmPHD6 improves stress tolerance in soybean (Glycinemax) plants. Knocking down the LHP1 expression disrupts this role of GmPHD6, indicating that GmPHD6 requires LHP1 functions during stress response. GmPHD6 influences expression of dozens of stress-related genes. Among these, we identified three targets of GmPHD6, including ABA-stress-ripening-induced CYP75B1 and CYP82C4 Overexpression of each gene confers stress tolerance in soybean plants. GmPHD6 is recruited to H3K4me0/1/2 marks and recognizes the G-rich elements in target gene promoters, whereas LHP1 activates expression of these targets. Our study reveals a mechanism involving two partners in a complex. Manipulation of the genes in this pathway should improve stress tolerance in soybean or other legumes/crops.

Ethylene-Inhibited Jasmonic Acid Biosynthesis Promotes Mesocotyl/Coleoptile Elongation of Etiolated Rice Seedlings.

Elongation of the mesocotyl and coleoptile facilitates the emergence of rice (Oryza sativa) seedlings from soil and is affected by various genetic and environment factors. The regulatory mechanism underlying this process remains largely unclear. Here, we examined the regulation of mesocotyl and coleoptile growth by characterizing a gaoyao1 (gy1) mutant that exhibits a longer mesocotyl and longer coleoptile than its original variety of rice. GY1 was identified through map-based cloning and encodes a PLA1-type phospholipase that localizes in chloroplasts. GY1 functions at the initial step of jasmonic acid (JA) biosynthesis to repress mesocotyl and coleoptile elongation in etiolated rice seedlings. Ethylene inhibits the expression of GY1 and other genes in the JA biosynthesis pathway to reduce JA levels and enhance mesocotyl and coleoptile growth by promoting cell elongation. Genetically, GY1 acts downstream of the OsEIN2-mediated ethylene signaling pathway to regulate mesocotyl/coleoptile growth. Through analysis of the resequencing data from 3000 rice accessions, we identified a single natural variation of the GY1 gene, GY1376T , which contributes to mesocotyl elongation in rice varieties. Our study reveals novel insights into the regulatory mechanism of mesocotyl/coleoptile elongation and should have practical applications in rice breeding programs.