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Background The Northeast Plain of China, which is an important region for the production of high grain quality rice in China. However, the grain quality of the rice produced varies across this region, even for the same cultivar. Objective In order to explore the meteorological factors that have the greatest influence on quality and the transcriptional level differences between different cultivars and different locations at grain filling stage. Methods We grew eight rice cultivars in three locations in Northeast China during two growing seasons (2017 and 2018). We recorded meteorological conditions, including air temperature, air temperature range, and photosynthetically active radiation (PAR) during the grain-filling stage of each cultivar, and analyzed the grain quality of those eight cultivars. Results Across all eight cultivars, meteorological factors had a stronger effect on eating quality than genotype, while genotype had a stronger effect on milling quality. Of the three environmental factors assessed, PAR was significantly correlated with the most grain quality traits. Using RNA-sequencing analysis, we identified 573 environment-specific DEGs (Differentially Expressed Genes), and 119 genotype-specific DEGs; 11 DEGs were responsive to genotype × environment interactions. These DEGs were involved in many key metabolic processes. Conclusion Our results indicated that interactions among environmental factors, especially PAR, affected rice quality in Northeast China. Further analyses of the DEGs identified herein may provide useful information for future breeding programs aiming to develop high grain quality rice varieties suitable for cultivation across Northeast China.

Rice (Oryza sativa) is sensitive to low temperatures, which affects the yield and quality of rice. Therefore, uncovering the molecular mechanisms behind chilling tolerance is a critical task for improving cold tolerance in rice cultivars. Here, we report that OsWRKY63, a WRKY transcription factor with an unknown function, negatively regulates chilling tolerance in rice. OsWRKY63-overexpressing rice lines are more sensitive to cold stress. Conversely, OsWRKY63-knockout mutants generated using a CRISPR/Cas9 genome editing approach exhibited increased chilling tolerance. OsWRKY63 was expressed in all rice tissues, and OsWRKY63 expression was induced under cold stress, dehydration stress, high salinity stress, and ABA treatment. OsWRKY63 localized in the nucleus plays a role as a transcription repressor and downregulates many cold stress-related genes and reactive oxygen species scavenging-related genes. Molecular, biochemical, and genetic assays showed that OsWRKY76 is a direct target gene of OsWRKY63 and that its expression is suppressed by OsWRKY63. OsWRKY76-knockout lines had dramatically decreased cold tolerance, and the cold-induced expression of five OsDREB1 genes was repressed. OsWRKY76 interacted with OsbHLH148, transactivating the expression of OsDREB1B to enhance chilling tolerance in rice. Thus, our study suggests that OsWRKY63 negatively regulates chilling tolerance through the OsWRKY63-OsWRKY76-OsDREB1B transcriptional regulatory cascade in rice.

Arabidopsis cryptochrome 2 (CRY2) and FLAVIN-BINDING, KELCH REPEAT, and F-BOX 1 (FKF1) are blue light receptors mediating light regulation of growth and development, such as photoperiodic flowering. CRY2 interacts with a basic helix–loop–helix transcription factor CIB1 in response to blue light to activate the transcription of the flowering integrator gene FLOWERING LOCUS T (FT). CIB1, CIB2, CIB4, and CIB5 function redundantly to promote flowering in a CRY2-dependent way and form various heterodimers to bind to the noncanonical E-box sequence in the FT promoter. However, the function of CIB3 has not been described. We discovered that CIB3 promotes photoperiodic flowering independently of CRY2. Moreover, CIB3 does not interact with CRY2 but interacts with CIB1 and functions synergistically with CIB1 to promote the transcription of the GI gene. FKF1 is required for CIB3 to promote flowering and enhances the CIB1–CIB3 interaction in response to blue light.

WRKY transcription factors (TFs) have been reported to respond to biotic and abiotic stresses and regulate plant growth and development. However, the molecular mechanisms of WRKY TFs involved in drought stress and regulating plant height in rice remain largely unknown. In this study, we found that transgenic rice lines overexpressing OsWRKY55 (OsWRKY55-OE) exhibited reduced drought resistance. The OsWRKY55-OE lines showed faster water loss and greater accumulation of hydrogen peroxide (H2O2) and superoxide radical (O2−·) compared to wild-type (WT) plants under drought conditions. OsWRKY55 was expressed in various tissues and was induced by drought and abscisic acid (ABA) treatments. Through yeast two-hybrid assays, we found that OsWRKY55 interacted with four mitogen-activated protein kinases (MAPKs) that could be induced by drought, including OsMPK7, OsMPK9, OsMPK20-1, and OsMPK20-4. The activation effects of the four OsMPKs on OsWRKY55 transcriptional activity were demonstrated by a GAL4-dependent chimeric transactivation assay in rice protoplasts. Furthermore, OsWRKY55 was able to reduce plant height under normal conditions by decreasing the cell size. In addition, based on a dual luciferase reporter assay, OsWRKY55 was shown to bind to the promoter of OsAP2-39 through a yeast one-hybrid assay and positively regulate OsAP2-39 expression. These results suggest that OsWRKY55 plays a critical role in responses to drought stress and the regulation of plant height in rice, further providing valuable information for crop improvement.

The APETALA 2/ethylene response factors (AP2/ERF) are widespread in the plant kingdom and play essential roles in regulating plant growth and development as well as defense responses. In this study, a novel rice AP2/ERF transcription factor gene, OsRPH1, was isolated and functionally characterized. OsRPH1 falls into group-IVa of the AP2/ERF family. OsRPH1 protein was found to be localized in the nucleus and possessed transcriptional activity. Overexpression of OsRPH1 resulted in a decrease in plant height and length of internode and leaf sheath as well as other abnormal characters in rice. The length of the second leaf sheath of OsRPH1-overexpressing (OE) plants recovered to that of Kitaake (non-transgenic recipient) in response to exogenous gibberellin A3 (GA3) application. The expression of GA biosynthesis genes (OsGA20ox1-OsGA20ox4, OsGA3ox1, and OsGA3ox2) was significantly downregulated, whereas that of GA inactivation genes (OsGA2ox7, OsGA2ox9, and OsGA2ox10) was significantly upregulated in OsRPH1-OE plants. Endogenous bioactive GA contents significantly decreased in OsRPH1-OE plants. OsRPH1 interacted with a blue light receptor, OsCRY1b, in a blue light-dependent manner. Taken together, our results demonstrate that OsRPH1 negatively regulates plant height and bioactive GA content by controlling the expression of GA metabolism genes in rice. OsRPH1 is involved in blue light inhibition of leaf sheath elongation by interacting with OsCRY1b.

OVATE family proteins (OFPs) are plant-specific transcription factors that regulate plant growth and development. OFPs interact with 3-aa loop extension (TALE) homeodomain proteins and brassinosteroid (BR) signaling components to modulate gibberellic acid (GA) biosynthesis and BR responses. Bioactive GAs are essential in regulating plant organogenesis and organ growth by promoting cell differentiation and elongation. DELLA proteins act as the central repressors of GA-regulated processes and are targeted to be degraded by the 26S proteasome in the presence of GA. We discovered that the rice OFP22 negatively regulates GA and BR signal transduction. OsOFP22 expression was rapidly up-regulated by exogenous GA and BR application, detected predominantly in the calli and spikelets. Overexpression of OsOFP22 conferred multiple morphological phenotypes, including reduced plant height, dark green leaves, and shortened and widened leaves, floral organs and grains. The GA-induced elongation of the second leaf sheath in the seedlings, and α-amylase activity in the endosperms were attenuated in transgenic lines overexpressing OsOFP22, while GA-biosynthesis gene transcripts and bioactive GA3 and GA4 contents were increased in the transgenic plants. OsOFP22 promotes the protein accumulation of SLR1, the single DELLA in rice protein. Furthermore, Overexpression of OsOFP22 suppresses BR response and the expression of BR-related genes. OsOFP22 is thus involved in the repression of GA and BR signal transduction and integrates GA with BR to regulate plant growth and development.

Cryptochromes are blue/UV-A light receptors that mediate various aspects of plant growth and development. Here, we report the function and signal mechanism of cryptochrome 1b (SbCRY1b) from sweet sorghum [Sorghum bicolor (L.) Moench], a typical short-day cereal plant, to explore its potential for genetic improvement of sweet sorghum varieties. SbCRY1b mRNA enrichment showed almost 24-h diurnal rhythms in both short-day (SD) and long-day (LD) conditions. Overexpression of SbCRY1b rescued the late-flowering and the long hypocotyl phenotypes of cry1cry2 double mutant in the transgenic Arabidopsis. SbCRY1b mediated Arabidopsis FT mRNA expression in LD and HY5 protein accumulation in response to blue light. SbCRY1b protein was located in both the nucleus and cytoplasm and was degraded by 26S proteasomes in response to blue light. SbCRY1b interacted, respectively, with Arabidopsis suppressor of PHYA-1051 (AtSPA1), E3 ubiquitin ligase constitutive photomorphogenesis 1 (AtCOP1), and a putative COP1 from sweet sorghum (SbCOP1) instead of SbSPA1 in vitro in a blue light-dependent manner. The observations imply SbCRY1b functions as a major regulator of photoperiodic flowering and its function is more similar to that of Arabidopsis CRY2. Moreover, SbCRY1b-overexpressed transgenic Arabidopsis showed oversensitivity to abscisic acid (ABA) during seed germination and root development. The expression of abscisic acid-insensitive 4 (ABI4), ABI5, abscisic acid responsive element-binding 1 (ABF1), (sucrose non-fermenting 1)-related protein kinase (SnRK2.3), RD29A, and EM6 was upregulated in the transgenic Arabidopsis. The results demonstrated that SbCRY1b may integrate blue light and ABA signals to regulate plant development.

This work provides the bioinformatics, expression pattern and functional analyses of cryptochrome 1a from sweet sorghum (SbCRY1a), together with an exploration of the signaling mechanism mediated by SbCRY1a. Sweet sorghum [Sorghum bicolor (L.) Moench] is considered to be an ideal candidate for biofuel production due to its high efficiency of photosynthesis and the ability to maintain yield under harsh environmental conditions. Blue light receptor cryptochromes regulate multiple aspects of plant growth and development. Here, we reported the function and signal mechanism of sweet sorghum cryptochrome 1a (SbCRY1a) to explore its potential for genetic improvement of sweet sorghum varieties. SbCRY1a transcripts experienced almost 24 h diurnal cycling; however, its protein abundance showed no oscillation. Overexpression of SbCRY1a in Arabidopsis rescued the phenotype of cry1 mutant in a blue light-specific manner and regulated HY5 accumulation under blue light. SbCRY1a protein was present in both nucleus and cytoplasm. The photoexcited SbCRY1a interacted directly with a putative RING E3 ubiquitin ligase constitutive photomorphogenesis 1 (COP1) from sweet sorghum (SbCOP1) instead of SbSPA1 to suppress SbCOP1–SbHY5 interaction responding to blue light. These observations indicate that the function and signaling mechanism of cryptochromes are basically conservative between monocotyledons and dicotyledons. Moreover, SbCRY1a-overexpressed transgenic Arabidopsis showed oversensitive to abscisic acid (ABA) and salinity. The ABA-responsive gene ABI5 was up-regulated evidently in SbCRY1a transgenic lines, suggesting that SbCRY1a might regulate ABA signaling through the HY5-ABI5 regulon.

Aluminium (Al) toxicity is a primary limitation of crop production in acid soils, which take over 40% of arable soil worldwide. In previous studies, a series of genes have been identified to regulate the plant Al resistance or tolerance. However, none of E3 ubiquitin ligase, the key factor of ubiquitination that plays an important role in plant growth and development, has been characterized for Al response in Arabidopsis. In this study, an E3 ubiquition ligase gene AtATRF1 (Al Tolerance RING Finger 1), a homolog of RAD18 interacting RAD6 to repair the damaged DNA in human and yeast, is isolated from Arabidopsis. It encodes a predicted protein of 296 amino acids with a C3HC4 type RING finger domain. The expression of AtATRF1 is induced by Al, and the transgenic plant overexpressing AtATRF1 enhances the Al tolerance. Similar as RAD18, the AtATRF1 locates in nucleus and regulates the expression of AtATR, which involves in DNA repair and Al response in Arabidopsis. Our results indicate that nuclearlocated AtATRF1 may interact and ubiquitinate the transcriptional regulator of AtATR to mediate the Al tolerance of Arabidopsis.

Cryptochromes are blue light receptors regulated by light-dependent ubiquitination and degradation in both plant and animal lineages. The Arabidopsis genome encodes two cryptochromes, CRY1 and CRY2, of which CRY2 undergoes blue light-dependent ubiquitination and 26S proteasome-dependent degradation. The molecular mechanism regulating blue light-dependent proteolysis of CRY2 is still not fully understood. We found that the F-box proteins ZEITLUPE (ZTL) and Lov Kelch Protein2 (LKP2), which mediate blue light suppression of degradation of the CRY2 signaling partner CIB1, are not required for the blue light-dependent CRY2 degradation. We further showed that the previously reported function of the COP1–SPA1 protein complex in blue light-dependent CRY2 degradation is more likely to be attributable to its cullin 4 (CUL4)-based E3 ubiquitin ligase activity than its activity as the cryptochrome signaling partner. However, the blue light-dependent CRY2 degradation is only partially impaired in the cul4 mutant, the cop1-5 null mutant and the spa1234 quadruple mutant, suggesting a possible involvement of additional E3 ubiquitin ligases in the regulation of CRY2. Consistent with this hypothesis, we demonstrated that the blue light-dependent CRY2 degradation is significantly impaired in the temperature-sensitive cul1 mutant allele (axr6-3), especially under the non-permissive temperature. Based on these and other results presented, we propose that photoexcited CRY2 undergoes Lys48-linked polyubiquitination catalyzed by the CUL4- and CUL1-based E3 ubiquitin ligases.

In this study, we constructed dual-transgene vectors (pDT1, pDT7, and pDT7G) that simultaneously co-expressed two genes in plants. ACTIN2 and UBQ10 promoters were used to control the expression of these two genes. The 4×Myc, 3×HA, and 3×Flag reporter genes allowed for the convenient identification of a tunable co-expression system in plants, whereas the dexamethasone (Dex) inducible reporter gene C-terminus of the glucocorticoid receptor (cGR) provided Dex-dependent translocation of the fusion gene between the nucleus and cytoplasm. The function of pDT vectors was validated using four pairwise genes in Nicotiana benthamiana or Arabidopsis thaliana. The co-expression efficiency of two genes from the pDT1 and pDT7G vectors was 35% and 42%, respectively, which ensured the generation of sufficient transgenic materials. These pDT vectors are simple, reliable, efficient, and time-saving tools for the co-expression of two genes through a single transformation event and can be used in the study of protein-protein interactions or multi-component complexes.

Protein tyrosine phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. However, the cellular roles for tyrosine phosphorylation in plant remain unclear. Here we describe that tyrosine phosphorylation involved in stomata closing pathway. Genistein, an inhibitor of protein tyrosine kinases (PTKs), could prevent darkness and abscisic acid (ABA)-induced stomatal closure of Vicia faba L. Stomatal closure induced by high external Ca2+ was prevented by phenylarsine oxide (PAO), a specific inhibitor of protein tyrosine phosphatases (PTPs), which promoted closed stomata reopening. Using anti-phosphotyrosine antibody, we detected the distribution of phosphotyrosyl proteins (PYP) by the immuno-fluorescence and immuno-gold electron microscope techniques in guard cells of Vicia faba. It shows that PYP is localized to nucleus, cytoplasm, vacuole and chloroplast of guard cells. Interestingly, the distribution of PYP in chloroplast of guard cell mainly was localized on the surface of starch granule, indicating that there is some relationship between tyrosine phosphorylation and starch metabolism in guard cells. In addition, the starch content in leaves of Vicia faba can be enhanced by inhibiting the activity of PTPs with PAO. Overall, these studies and combined previous report suggest that tyrosine phosphorylation may modulate starch degradation in guard cells, and thus regulating the stomatal movement.

The objective was to study the relationship between maize hybrids and their parents in potassium ion (K+)-uptake characteristics under the stress from a low amount of potassium. Superoxide dismutase (SOD) activity, the malondialdehyde (MDA) content, the potassium utilization index (KUI), root volume, total and active root uptake area, Michaelis-Menten coefficient (Km), and the maximum rate of uptake (Vm) of different maize (Zea mays L.) genotypes were determined. SOD activity and MDA content in the leaves of Si144, Xianyu335 and PH4CV were significantly higher than the control, whereas for Jidan27, Si-287 and PH6WC, no differences were detected. Jidan27 showed a higher KUI than Xianyu335 under 0.1 mM potassium chloride (KCl), but no differences were observed under 3 mM KCl. Jidan27, Si287, and PH6WC had a greater root volume, total and active uptake areas, higher Vm and smaller Km than the other genotypes under 0.1 mM KCl. The differences in the KUI among maize genotypes resulted from variation in the Km ...

Alternative splicing (AS), i.e., generating multiple mRNA transcripts from a single gene, is known to contribute to proteome diversity and gene regulation in eukaryotes. OsHsfA2a, a gene encoding a heat shock transcription factor (HSF) in rice (Oryza. sativa L. cv. Nipponbare) generates six transcript isoforms (OsHsfA2a-1 to OsHsfA2a-6) by AS. In this study, an OsHsfA2a has been identified and the genomic sequence found to be composed of three exons and two introns. Moreover, stress-responsive and phytohormone-responsive cis-elements in the promoter regions of OsHsfA2a have been identified. Real-time quantitative polymerase chain reaction analysis has revealed that the six isoforms have different tissue-specific expression patterns under normal growth conditions in rice. Interestingly, when treated with ABA (abscisic acid) and various abiotic stresses such as heat shock, simulated drought and high NaCl, these isoforms also show various stress responsive patterns in rice seedlings. These results indicate that AS of OsHsfA2a may play an important role not only in rice growth and development, but also in regulating stress-specific cellular adaptation responses.

Author(s): Zhao, Tao; Liu, Jun; Li, Hong-Yu; Lin, Jian-Zhong; Bian, Ming-Di; Zhang, Chun-Yu; Zhang, Yong-Xing; Peng, Yu-Chong; Liu, Bin; Lin, ChenTao

© 2015, National Academy of Sciences. All rights reserved. Cryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photo-reduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the "Trp triad." It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1W324Aand CRY1W400Fmutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.

© 2015 The Author. Arabidopsis cryptochrome 2 (CRY2) is a blue light receptor that mediates light inhibition of hypocotyl elongation and long-day promotion of floral initiation. CRY2 is known to undergo blue light-dependent phosphorylation, which is believed to serve regulatory roles in the function of CRY2. We report here on a biochemical and genetics study of CRY2 phosphorylation. Using mass spectrometry analysis, we identified three serine residues in the CCE domain of CRY2 (S588, S599, and S605) that undergo blue light-dependent phosphorylation in Arabidopsis seedlings. A study of serine-substitution mutations in the CCE domain of CRY2 demonstrates that CRY2 contains two types of phosphorylation in the CCE domain, one in the serine cluster that causes electrophoretic mobility upshift and the other outside the serine cluster that does not seem to cause mobility upshift. We showed that mutations in the serine residues within and outside the serine cluster diminished blue light-dependent CRY2 phosphorylation, degradation, and physiological activities. These results support the hypothesis that blue light-dependent phosphorylation of the CCE domain determines the photosensitivity of Arabidopsis CRY2.