Zhi-Yong Li

Isolation and functional characterization of HvDREB1 : a gene encoding a dehydration-responsive element binding protein in Hordeum vulgare

A gene encoding Hordeum vulgare dehydration-responsive element binding protein 1 (HvDREB1), a member of the A-2 subgroup of the DREB subfamily, was isolated from barley seedlings. A subcellular localization assay revealed accumulation of HvDREB1 protein in the nucleus. As a trans-acting factor, HvDREB1 was able to bind to DRE/CRT elements and transactivate reporter gene expression in yeast cells. A study of various deletion mutants of HvDREB1 proteins indicated that the transactivation activity was localized to the N-terminal region. Expression of the HvDREB1 gene in barley leaves was significantly induced by salt, drought, and low-temperature. In contrast to most A-2 subgroup members in Arabidopsis thaliana, HvDREB1 also responded to exogenous ABA. Overexpression of HvDREB1 activated a downstream gene, RD29A, under normal growth conditions and led to increased tolerance to salt stress in Arabidopsis plants. These results suggest that HvDREB1 produces a DRE-/CRT-binding transcription factor that may have an important role in improving salt tolerance in plants.

Overexpression of soybean GmCBL1 enhances abiotic stress tolerance and promotes hypocotyl elongation in Arabidopsis.

Although extensive studies and remarkable progress have been made with Arabidopsis calcineurin B-like proteins (CBLs), knowledge of their functions in other plant species is still limited. Here we isolated gene GmCBL1 from soybean, a homolog of AtCBL1 in Arabidopsis. GmCBL1 was differentially induced by multiple abiotic stress and plant hormones, and its transcripts were abundant in seedlings and mature roots. We over-expressed GmCBL1 in Arabidopsis and found that it enhanced tolerances to both high salt and drought stresses in the transgenic plants. Overexpression of GmCBL1 also promoted hypocotyl elongation under light conditions. GmCBL1 may regulate stress tolerance through activation of stress-related genes, and may control hypocotyl development by altering the expression of gibberellin biosynthesis-related genes. This study identifies a putative soybean CBL gene that functions in both stress tolerance and light-dependent hypocotyl development.

A mutation in Arabidopsis BSK5 encoding a brassinosteroid-signaling kinase protein affects responses to salinity and abscisic acid

As the most recently characterized group of plant hormones, brassinosteroids (BR) are involved in a number of physiological responses. Although many key components of the BR signaling pathway have been isolated and characterized, there is little information on detailed characterization of brassinosteroid-signaling kinase (BSK) proteins. In this study, Arabidopsis BSK5 was isolated and functionally analyzed. BSK5 transcripts were detected in various tissues, and were induced by abiotic stresses including salt and drought, as well as phytohormones of BR and abscisic acid (ABA). Arabidopsis loss-of-function mutant bsk5 exhibited sensitivity to salinity and ABA. Mutations of the BSK5 gene also altered the expression of several stress-regulated genes. We suggest that BSK5 responds to other signals as well as BR.

Deregulation of the OsmiR160 Target Gene OsARF18 Causes Growth and Developmental Defects with an Alteration of Auxin Signaling in Rice

MicroRNAs (miRNAs) control gene expression as key negative regulators at the post-transcriptional level. MiR160 plays a pivotal role in Arabidopsis growth and development through repressing expression of its target AUXIN RESPONSE FACTOR (ARF) genes; however, the function of miR160 in monocots remains elusive. In this study, we found that the mature rice miR160 (OsmiR160) was mainly derived from OsMIR160a and OsMIR160b genes. Among four potential OsmiR160 target OsARF genes, the OsARF18 transcript was cleaved at the OsmiR160 target site. Rice transgenic plants (named mOsARF18) expressing an OsmiR160-resistant version of OsARF18 exhibited pleiotropic defects in growth and development, including dwarf stature, rolled leaves, and small seeds. mOsARF18 leaves were abnormal in bulliform cell differentiation and epidermal cell division. Starch accumulation in mOsARF18 seeds was also reduced. Moreover, auxin induced expression of OsMIR160a, OsMIR160b, and OsARF18, whereas expression of OsMIR160a and OsMIR160b as well as genes involved in auxin signaling was altered in mOsARF18 plants. Our results show that negative regulation of OsARF18 expression by OsmiR160 is critical for rice growth and development via affecting auxin signaling, which will advance future studies on the molecular mechanism by which miR160 fine-tunes auxin signaling in plants.

A novel role for Arabidopsis CBL1 in affecting plant responses to glucose and gibberellin during germination and seedling development.

Glucose and phytohormones such as abscisic acid (ABA), ethylene, and gibberellin (GA) coordinately regulate germination and seedling development. However, there is still inadequate evidence to link their molecular roles in affecting plant responses. Calcium acts as a second messenger in a diverse range of signal transduction pathways. As calcium sensors unique to plants, calcineurin B-like (CBL) proteins are well known to modulate abiotic stress responses. In this study, it was found that CBL1 was induced by glucose in Arabidopsis. Loss-of-function mutant cbl1 exhibited hypersensitivity to glucose and paclobutrazol, a GA biosynthetic inhibitor. Several sugar-responsive and GA biosynthetic gene expressions were altered in the cbl1 mutant. CBL1 protein physically interacted with AKINβ1, the regulatory β subunit of the SnRK1 complex which has a central role in sugar signaling. Our results indicate a novel role for CBL1 in modulating responses to glucose and GA signals.

Origin and Development of the Root Cap in Rice

Cellular and molecular events are inherent to the formation and development of the root cap in rice. The tip of the root is covered by a thimble-shaped root cap that is the site of perception and transduction for many environmental stimuli. Until now, little was known about how the root cap of rice (Oryza sativa) develops and functions to regulate the adaptive behavior of the root. To address this, we examined the formation of the rice root cap during embryogenesis and characterized the anatomy and structure of the rice radicle root cap. We further investigated the role of the quiescent center in the de novo origin of the root cap. At the molecular level, we found that shoot-derived auxin was absolutely needed to trigger root cap regeneration when the quiescent center was removed. Our time-course analysis of transcriptomic dynamics during the early phases of root cap regeneration indicated that changes in auxin signaling and appropriate levels of cytokinin are critical for root cap regeneration after the removal of the root cap. Moreover, we identified 152 genes that produce root cap-specific transcripts in the rice root tip. These findings together offer, to our knowledge, new mechanistic insights into the cellular and molecular events inherent in the formation and development of the root cap in rice and provide a basis for future research on the developmental and physiological function of the root cap of monocot crops.

The Voltage-Dependent Anion Channel 1 (AtVDAC1) Negatively Regulates Plant Cold Responses during Germination and Seedling Development in Arabidopsis and Interacts with Calcium Sensor CBL1.

The voltage-dependent anion channel (VDAC), a highly conserved major mitochondrial outer membrane protein, plays crucial roles in energy metabolism and metabolite transport. However, knowledge about the roles of the VDAC family in plants is limited. In this study, we investigated the expression pattern of VDAC1 in Arabidopsis and found that cold stress promoted the accumulation of VDAC1 transcripts in imbibed seeds and mature plants. Overexpression of VDAC1 reduced tolerance to cold stress in Arabidopsis. Phenotype analysis of VDAC1 T-DNA insertion mutant plants indicated that a vdac1 mutant line had faster germination kinetics under cold treatment and showed enhanced tolerance to freezing. The yeast two-hybrid system revealed that VDAC1 interacts with CBL1, a calcium sensor in plants. Like the vdac1, a cbl1 mutant also exhibited a higher seed germination rate. We conclude that both VDAC1 and CBL1 regulate cold stress responses during seed germination and plant development.

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

The Leu-rich repeat receptor-like kinases SERK1 and SERK2 affect anther cell differentiation as a partners of EMS1 in Arabidopsis. Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

Carbonic Anhydrases Function in Anther Cell Differentiation Downstream of the Receptor-like Kinase EMS1

β-Carbonic anhydrases are posttranslationally modified by EMS1 and act as direct downstream effectors of this receptor-like kinase, highlighting their crucial role in cell differentiation. Plants extensively employ leucine-rich repeat receptor-like kinases (LRR-RLKs), the largest family of RLKs, to control a wide range of growth and developmental processes as well as defense responses. To date, only a few direct downstream effectors for LRR-RLKs have been identified. We previously showed that the LRR-RLK EMS1 (EXCESS MICROSPOROCYTES1) and its ligand TPD1 (TAPETUM DETERMINANT1) are required for the differentiation of somatic tapetal cells and reproductive microsporocytes during early anther development in Arabidopsis thaliana. Here, we report the identification of β-carbonic anhydrases (βCAs) as the direct downstream targets of EMS1. EMS1 biochemically interacts with βCA proteins. Loss of function of βCA genes caused defective tapetal cell differentiation, while overexpression of βCA1 led to the formation of extra tapetal cells. EMS1 phosphorylates βCA1 at four sites, resulting in increased βCA1 activity. Furthermore, phosphorylation-blocking mutations impaired the function of βCA1 in tapetal cell differentiation; however, a phosphorylation mimic mutation promoted the formation of tapetal cells. βCAs are also involved in pH regulation in tapetal cells. Our findings highlight the role of βCA in controlling cell differentiation and provide insights into the posttranslational modification of carbonic anhydrases via receptor-like kinase-mediated phosphorylation.