A forward genetic screen to explore chloroplast protein import in vivo identifies Moco sulfurase,pivotal for ABA and IAA biosynthesis and purine turnover

A genetic screen in Arabidopsis was developed to explore the regulation of chloroplast protein import in vivo using two independent reporters representing housekeeping and photosynthetic pre‐proteins. We first used 5‐enolpyruvylshikimate 3‐phosphate synthase (EPSP synthase*), a key enzyme in the shikimic acid pathway, with a mutation that confers tolerance to the herbicide glyphosate. Because the EPSP synthase* pre‐protein must be imported for its function, the loss of glyphosate tolerance provided an initial indication of an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5–GFP) was determined. A class of altered chloroplast import (aci) mutants showed both glyphosate sensitivity and FD5–GFP mislocalized to nuclei. aci2‐1 was selected for further study. Yellow fluorescent protein (YFP) fused to the transit peptide of EPSP synthase* or the small subunit of Rubisco was not imported into chloroplasts, but also localized to nuclei during protoplast transient expression. Isolated aci2‐1 chloroplasts showed a 50% reduction in pre‐protein import efficiency in an in vitro assay. Mutants did not grow photoautotrophically on media without sucrose and were small and dark green in soil. aci2‐1 and two alleles code for Moco‐sulfurase, which activates the aldehyde oxidases required for the biosynthesis of the plant hormones abscisic acid (ABA) and indole‐acetic acid (IAA) and controls purine nucleotide (ATP and GTP) turnover and nitrogen recycling via xanthine dehydrogenase. These enzyme activities were not detected in aci2‐1. ABA, IAA and/or purine turnover may play previously unrecognized roles in the regulation of chloroplast protein import in response to developmental, metabolic and environmental cues.     

LcFIN2, a novel chloroplast protein gene from sheepgrass,enhances tolerance to low temperature in Arabidopsis and rice

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA‐sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing‐induced protein 2, encoded a chloroplast‐targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non‐photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal‐related and photosynthesis‐related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low‐temperature tolerance in crops.     

Knockout of AtDjB1, a J‐domain protein from Arabidopsis thaliana,alters plant responses to osmotic stress and abscisic acid

AtDjB1 is a member of the Arabidopsis thaliana J‐protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1‐1) exhibited greater ABA sensitivity compared with the wild‐type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA‐responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1‐1 plants exhibited higher glucose levels and greater glucose sensitivity in the post‐germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1‐1 than WT seedlings. Taken together, higher glucose levels in atj1‐1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.     

OsDMI3‐mediated activation of OsMPK1 regulates the activities of antioxidant enzymes in abscisic acid signalling in rice

In rice, the Ca²⁺/calmodulin (CaM)‐dependent protein kinase (CCaMK) OsDMI3 has been shown to be required for abscisic acid (ABA)‐induced antioxidant defence. However, it is not clear how OsDMI3 participates in this process in rice. In this study, the cross‐talk between OsDMI3 and the major ABA‐activated MAPK OsMPK1 in ABA‐induced antioxidant defence was investigated. ABA treatment induced the expression of OsDMI3 and OsMPK1 and the activities of OsDMI3 and OsMPK1 in rice leaves. In the mutant of OsDMI3, the ABA‐induced increases in the expression and the activity of OsMPK1 were substantially reduced. But in the mutant of OsMPK1, the ABA‐induced increases in the expression and the activity of OsDMI3 were not affected. Pretreatments with MAPKK inhibitors also did not affect the ABA‐induced activation of OsDMI3. Further, a transient expression analysis in combination with mutant analysis in rice protoplasts showed that OsMPK1 is required for OsDMI3‐induced increases in the activities of antioxidant enzymes and the production of H₂O₂. Our data indicate that there exists a cross‐talk between OsDMI3 and OsMPK1 in ABA signalling, in which OsDMI3 functions upstream of OsMPK1 to regulate the activities of antioxidant enzymes and the production of H₂O₂ in rice.     

OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2 signalling in rice

Drought is one of the major abiotic stresses that directly implicate plant growth and crop productivity. Although many genes in response to drought stress have been identified, genetic improvement to drought resistance especially in food crops is showing relatively slow progress worldwide. Here, we reported the isolation of abscisic acid, stress and ripening (ASR) genes from upland rice variety, IRAT109 (Oryza sativa L. ssp. japonica), and demonstrated that overexpression of OsASR5 enhanced osmotic tolerance in Escherichia coli and drought tolerance in Arabidopsis and rice by regulating leaf water status under drought stress conditions. Moreover, overexpression of OsASR5 in rice increased endogenous ABA level and showed hypersensitive to exogenous ABA treatment at both germination and postgermination stages. The production of H₂O₂, a second messenger for the induction of stomatal closure in response to ABA, was activated in overexpression plants under drought stress conditions, consequently, increased stomatal closure and decreased stomatal conductance. In contrast, the loss‐of‐function mutant, osasr5, showed sensitivity to drought stress with lower relative water content under drought stress conditions. Further studies demonstrated that OsASR5 functioned as chaperone‐like protein and interacted with stress‐related HSP40 and 2OG‐Fe (II) oxygenase domain containing proteins in yeast and plants. Taken together, we suggest that OsASR5 plays multiple roles in response to drought stress by regulating ABA biosynthesis, promoting stomatal closure, as well as acting as chaperone‐like protein that possibly prevents drought stress‐related proteins from inactivation.     

Suppression of DRR1 results in the accumulation of insoluble ubiquitinated proteins,which impairs drought stress tolerance

Drought stress has detrimental effects on plants. Although the abscisic acid (ABA)‐mediated drought response is well established, defensive mechanisms to cope with dehydration‐induced proteotoxicity have been rarely studied. DRR1 was identified as an Arabidopsis drought‐induced gene encoding an ER‐localized RING‐type E3 Ub ligase. Suppression of DRR1 markedly reduced tolerance to drought and proteotoxic stress without altering ABA‐mediated germination and stomatal movement. Proteotoxicity‐ and dehydration‐induced insoluble ubiquitinated protein accumulation was more obvious in DRR1 loss‐of‐function plants than in wild‐type plants. These results suggest that DRR1 is involved in an ABA‐independent drought stress response possibly through the mitigation of dehydration‐induced proteotoxic stress.     

CLD1/SRL1 modulates leaf rolling by affecting cell wall formation,epidermis integrity and water homeostasis in rice

Leaf rolling is considered as one of the most important agronomic traits in rice breeding. It has been previously reported that SEMI‐ROLLED LEAF 1 (SRL1) modulates leaf rolling by regulating the formation of bulliform cells in rice (Oryza sativa); however, the regulatory mechanism underlying SRL1 has yet to be further elucidated. Here, we report the functional characterization of a novel leaf‐rolling mutant, curled leaf and dwarf 1 (cld1), with multiple morphological defects. Map‐based cloning revealed that CLD1 is allelic with SRL1, and loses function in cld1 through DNA methylation. CLD1/SRL1 encodes a glycophosphatidylinositol (GPI)‐anchored membrane protein that modulates leaf rolling and other aspects of rice growth and development. The cld1 mutant exhibits significant decreases in cellulose and lignin contents in secondary cell walls of leaves, indicating that the loss of function of CLD1/SRL1 affects cell wall formation. Furthermore, the loss of CLD1/SRL1 function leads to defective leaf epidermis such as bulliform‐like epidermal cells. The defects in leaf epidermis decrease the water‐retaining capacity and lead to water deficits in cld1 leaves, which contribute to the main cause of leaf rolling. As a result of the more rapid water loss and lower water content in leaves, cld1 exhibits reduced drought tolerance. Accordingly, the loss of CLD1/SRL1 function causes abnormal expression of genes and proteins associated with cell wall formation, cuticle development and water stress. Taken together, these findings suggest that the functional roles of CLD1/SRL1 in leaf‐rolling regulation are closely related to the maintenance of cell wall formation, epidermal integrity and water homeostasis.     

Pepper protein phosphatase type 2C,CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response

Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum A BA and D rought‐I nduced P rotein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1‐OX) exhibited an ABA‐hyposensitive and drought‐susceptible phenotype. We used a yeast two‐hybrid screening assay to identify CaRLP1 (Capsicum annuum R CAR‐L ike P rotein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1‐OX plants, CaRLP1‐OX plants displayed an ABA‐hypersensitive and drought‐tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress‐responsive genes relative to those of wild‐type plants. In CaADIP1‐OX/CaRLP1‐OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1‐induced ABA hyposensitivity during the germinative and post‐germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA‐dependent defense signalling response to drought stress.     

LALF32‐51‐E7, a HPV‐16 therapeutic vaccine candidate,forms protein body‐like structures when expressed in Nicotiana benthamiana leaves

High‐risk human papillomaviruses (HPVs) cause cervical cancer, and while there are good prophylactic vaccines on the market, these are ineffective against established infections, creating a clear need for therapeutic vaccines. The HPV E7 protein is one of the essential oncoproteins for the onset and maintenance of malignancy and is therefore an ideal therapeutic vaccine target. We fused the HPV‐16 E7 protein to the Limulus polyphemus antilipopolysaccharide factor (LALF_32‐51), a small hydrophobic peptide that can penetrate cell membranes and that has immunomodulatory properties. LALF_32‐51‐E7 was transiently expressed in Nicotiana benthamiana, and we previously determined that it accumulated better when targeted to chloroplasts compared to being localized in the cytoplasm. Subsequently, we aimed to prove whether LALF_32‐51‐E7 was indeed associated with the chloroplasts by determining its subcellular localization. The LALF_32‐51‐E7 gene was fused to one encoding enhanced GFP to generate a LG fusion protein, and localization was determined by confocal laser scanning microscopy and transmission electron microscopy (TEM). The fluorescence observed from chloroplast‐targeted LG was distinctively different from that of the cytoplasmic LG. Small spherical structures resembling protein bodies (PBs) were seen that clearly localized with the chloroplasts. Larger but less abundant PB‐like structures were also seen for the cytoplasmic LG. PB‐like structure formation was confirmed for both LG and LALF_32‐51‐E7 by TEM. LALF_32‐51‐E7 was indeed targeted to the chloroplasts by the chloroplast transit peptide used in this study, and it formed aggregated PB‐like structures. This study could open a new avenue for the use of LALF_32‐51 as a PB‐inducing peptide.     

Occurrence and characterization of PEND proteins in angiosperms

The PEND protein is a DNA-binding protein in the inner envelope membrane of the developing chloroplast. It consists of a short pre-sequence, an N-terminal DNA-binding domain (cbZIP), a central repeat domain, and a C-terminal transmembrane domain. PEND homologs have been detected in various angiosperms, including Arabidopsis thaliana, Brassica napus, Medicago truncatula, cucumber and cherry. Monocot homologs have also been detected in barley and rice, but sequence conservation was low in monocots. PEND-related sequences have not been detected in non-flowering plants and algae. Green fluorescent protein fusions consisting of the N-terminal as well as full-length PEND homologs in A. thaliana and B. napus were targeted to chloroplasts, and localized to nucleoids and chloroplast periphery, respectively. Immunoblot analysis suggested that crucifer homologs were present in chloroplasts probably as a dimer, as in the case of pea. These results suggest that PEND protein is present in angiosperms, and the homologs in crucifers are functionally analogous to the PEND protein in pea.