Expression of the CCCH‐tandem zinc finger protein gene OsTZF5 under a stress‐inducible promoter mitigates the effect of drought stress on rice grain yield under field conditions

Increasing drought resistance without sacrificing grain yield remains an ongoing challenge in crop improvement. In this study, we report that O ryza s ativa CCCH‐t andem z inc f inger protein 5 (OsTZF5) can confer drought resistance and increase grain yield in transgenic rice plants. Expression of OsTZF5 was induced by abscisic acid, dehydration and cold stress. Upon stress, OsTZF5‐GFP localized to the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF5 under the constitutive maize ubiquitin promoter exhibited improved survival under drought but also growth retardation. By introducing OsTZF5 behind the stress‐responsive OsNAC6 promoter in two commercial upland cultivars, Curinga and NERICA4, we obtained transgenic plants that showed no growth retardation. Moreover, these plants exhibited significantly increased grain yield compared to non‐transgenic cultivars in different confined field drought environments. Physiological analysis indicated that OsTZF5 promoted both drought tolerance and drought avoidance. Collectively, our results provide strong evidence that OsTZF5 is a useful biotechnological tool to minimize yield losses in rice grown under drought conditions.     

Expression of the Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) improves the shoot biomass of transgenic barley and increases grain yield in a saline field

Cereal varieties with improved salinity tolerance are needed to achieve profitable grain yields in saline soils. The expression of AVP1, an Arabidopsis gene encoding a vacuolar proton pumping pyrophosphatase (H⁺‐PPase), has been shown to improve the salinity tolerance of transgenic plants in greenhouse conditions. However, the potential for this gene to improve the grain yield of cereal crops in a saline field has yet to be evaluated. Recent advances in high‐throughput nondestructive phenotyping technologies also offer an opportunity to quantitatively evaluate the growth of transgenic plants under abiotic stress through time. In this study, the growth of transgenic barley expressing AVP1 was evaluated under saline conditions in a pot experiment using nondestructive plant imaging and in a saline field trial. Greenhouse‐grown transgenic barley expressing AVP1 produced a larger shoot biomass compared to null segregants, as determined by an increase in projected shoot area, when grown in soil with 150 mm NaCl. This increase in shoot biomass of transgenic AVP1 barley occurred from an early growth stage and also in nonsaline conditions. In a saline field, the transgenic barley expressing AVP1 also showed an increase in shoot biomass and, importantly, produced a greater grain yield per plant compared to wild‐type plants. Interestingly, the expression of AVP1 did not alter barley leaf sodium concentrations in either greenhouse‐ or field‐grown plants. This study validates our greenhouse‐based experiments and indicates that transgenic barley expressing AVP1 is a promising option for increasing cereal crop productivity in saline fields.     

Down‐regulation of CBP80 gene expression as a strategy to engineer a drought‐tolerant potato

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down‐regulation of nuclear cap‐binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA‐hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water‐deficit tolerance of crop plants.     

The genetic basis of drought tolerance in the high oil crop Sesamum indicum

Unlike most of the important food crops, sesame can survive drought but severe and repeated drought episodes, especially occurring during the reproductive stage, significantly curtail the productivity of this high oil crop. Genome‐wide association study was conducted for traits related to drought tolerance using 400 diverse sesame accessions, including landraces and modern cultivars. Ten stable QTLs explaining more than 40% of the phenotypic variation and located on four linkage groups were significantly associated with drought tolerance related traits. Accessions from the tropical area harboured higher numbers of drought tolerance alleles at the peak loci and were found to be more tolerant than those from the northern area, indicating a long‐term genetic adaptation to drought‐prone environments. We found that sesame has already fixed important alleles conferring survival to drought which may explain its relative high drought tolerance. However, most of the alleles crucial for productivity and yield maintenance under drought conditions are far from been fixed. This study also revealed that pyramiding the favourable alleles observed at the peak loci is of high potential for enhancing drought tolerance in sesame. In addition, our results highlighted two important pleiotropic QTLs harbouring known and unreported drought tolerance genes such as SiABI4, SiTTM3, SiGOLS1, SiNIMIN1 and SiSAM. By integrating candidate gene association study, gene expression and transgenic experiments, we demonstrated that SiSAM confers drought tolerance by modulating polyamine levels and ROS homeostasis, and a missense mutation in the coding region partly contributes to the natural variation of drought tolerance in sesame.     

Development and evaluation of novel salt‐tolerant Eucalyptus trees by molecular breeding using an RNA‐Binding‐Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.)

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA‐Binding‐Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened‐house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants’ acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high‐stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non‐transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate‐stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non‐transgenic status survived the 70 mM NaCl treatment, but after 6‐month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non‐transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non‐transgenic plants after 15‐week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.     

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.     

Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods

Future genetic improvement of sugarcane depends, in part, on the ability to produce high‐yielding transgenic cultivars with improved traits such as herbicide and insect resistance. Here, transgenic sugarcane plants generated by different transformation methods were assessed for field performance over 3 years. Agrobacterium‐mediated (Agro) transgenic events (35) were produced using four different Agrobacterium tumefaciens strains, while biolistic (Biol) transgenic events (48) were produced using either minimal linearized DNA (LDNA) transgene cassettes with 5′, 3′ or blunt ends or whole circular plasmid (PDNA) vectors containing the same transgenes. A combined analysis showed a reduction in growth and cane yield in Biol, Agro as well as untransformed tissue culture (TC) events, compared with the parent clone (PC) Q117 (no transformation or tissue culture) in the plant, first ratoon and second ratoon crops. However, when individual events were analysed separately, yields of some transgenic events from both Agro and Biol were comparable to PC, suggesting that either transformation method can produce commercially suitable clones. Interestingly, a greater percentage of Biol transformants were similar to PC for growth and yield than Agro clones. Crop ratoonability and sugar yield components (Brix%, Pol%, and commercial cane sugar (CCS)) were unaffected by transformation or tissue culture. Transgene expression remained stable over different crop cycles and increased with plant maturity. Transgene copy number did not influence transgene expression, and both transformation methods produced low transgene copy number events. No consistent pattern of genetic changes was detected in the test population using three DNA fingerprinting techniques.     

OsSUV3 dual helicase functions in salinity stress tolerance by maintaining photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. IR64)

To overcome the salinity‐induced loss of crop yield, a salinity‐tolerant trait is required. The SUV3 helicase is involved in the regulation of RNA surveillance and turnover in mitochondria, but the helicase activity of plant SUV3 and its role in abiotic stress tolerance have not been reported so far. Here we report that the Oryza sativa (rice) SUV3 protein exhibits DNA and RNA helicase, and ATPase activities. Furthermore, we report that SUV3 is induced in rice seedlings in response to high levels of salt. Its expression, driven by a constitutive cauliflower mosaic virus 35S promoter in IR64 transgenic rice plants, confers salinity tolerance. The T₁ and T₂ sense transgenic lines showed tolerance to high salinity and fully matured without any loss in yields. The T₂ transgenic lines also showed tolerance to drought stress. These results suggest that the introduced trait is functional and stable in transgenic rice plants. The rice SUV3 sense transgenic lines showed lesser lipid peroxidation, electrolyte leakage and H₂O₂ production, along with higher activities of antioxidant enzymes under salinity stress, as compared with wild type, vector control and antisense transgenic lines. These results suggest the existence of an efficient antioxidant defence system to cope with salinity‐induced oxidative damage. Overall, this study reports that plant SUV3 exhibits DNA and RNA helicase and ATPase activities, and provides direct evidence of its function in imparting salinity stress tolerance without yield loss. The possible mechanism could be that OsSUV3 helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in transgenic rice.     

Expression of an Arabidopsis molybdenum cofactor sulphurase gene in soybean enhances drought tolerance and increases yield under field conditions

LOS5/ABA3 gene encoding molybdenum cofactor sulphurase is involved in aldehyde oxidase (AO) activity in Arabidopsis, which indirectly regulates ABA biosynthesis and increased stress tolerance. Here, we used a constitutive super promoter to drive LOS5/ABA3 overexpression in soybean (Glycine max L.) to enhance drought tolerance in growth chamber and field conditions. Expression of LOS5/ABA3 was up‐regulated by drought stress, which led to increasing AO activity and then a notable increase in ABA accumulation. Transgenic soybean under drought stress had reduced water loss by decreased stomatal aperture size and transpiration rate, which alleviated leaf wilting and maintained higher relative water content. Exposed to drought stress, transgenic soybean exhibited reduced cell membrane damage by reducing electrolyte leakage and production of malondialdehyde and promoting proline accumulation and antioxidant enzyme activities. Also, overexpression of LOS5/ABA3 enhanced expression of stress‐up‐regulated genes. Furthermore, the seed yield of transgenic plants is at least 21% higher than that of wide‐type plants under drought stress conditions in the field. These data suggest that overexpression of LOS5/ABA3 could improve drought tolerance in transgenic soybean via enhanced ABA accumulation, which could activate expression of stress‐up‐regulated genes and cause a series of physiological and biochemical resistant responses.     

Overexpression of ubiquitin‐like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin‐like post‐translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate‐controlled growth chambers. Here, pasture‐type and turf‐type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.     

Abiotic stress QTL in lettuce crop–wild hybrids: comparing greenhouse and field experiments

The development of stress‐tolerant crops is an increasingly important goal of current crop breeding. A higher abiotic stress tolerance could increase the probability of introgression of genes from crops to wild relatives. This is particularly relevant to the discussion on the risks of new GM crops that may be engineered to increase abiotic stress resistance. We investigated abiotic stress QTL in greenhouse and field experiments in which we subjected recombinant inbred lines from a cross between cultivated Lactuca sativa cv. Salinas and its wild relative L. serriola to drought, low nutrients, salt stress, and aboveground competition. Aboveground biomass at the end of the rosette stage was used as a proxy for the performance of plants under a particular stress. We detected a mosaic of abiotic stress QTL over the entire genome with little overlap between QTL from different stresses. The two QTL clusters that were identified reflected general growth rather than specific stress responses and colocated with clusters found in earlier studies for leaf shape and flowering time. Genetic correlations across treatments were often higher among different stress treatments within the same experiment (greenhouse or field), than among the same type of stress applied in different experiments. Moreover, the effects of the field stress treatments were more correlated with those of the greenhouse competition treatments than to those of the other greenhouse stress experiments, suggesting that competition rather than abiotic stress is a major factor in the field. In conclusion, the introgression risk of stress tolerance (trans‐)genes under field conditions cannot easily be predicted based on genomic background selection patterns from controlled QTL experiments in greenhouses, especially field data will be needed to assess potential (negative) ecological effects of introgression of these transgenes into wild relatives.     

A petal‐specific InMYB1 promoter from Japanese morning glory: a useful tool for molecular breeding of floricultural crops

Production of novel transgenic floricultural crops with altered petal properties requires transgenes that confer a useful trait and petal‐specific promoters. Several promoters have been shown to control transgenes in petals. However, all suffer from inherent drawbacks such as low petal specificity and restricted activity during the flowering stage. In addition, the promoters were not examined for their ability to confer petal‐specific expression in a wide range of plant species. Here, we report the promoter of InMYB1 from Japanese morning glory as a novel petal‐specific promoter for molecular breeding of floricultural crops. First, we produced stable InMYB1_1kb::GUS transgenic Arabidopsis and Eustoma plants and characterized spatial and temporal expression patterns under the control of the InMYB1 promoter by histochemical β‐glucuronidase (GUS) staining. GUS staining patterns were observed only in petals. This result showed that the InMYB1 promoter functions as a petal‐specific promoter. Second, we transiently introduced the InMYB1_1 kb::GUS construct into Eustoma, chrysanthemum, carnation, Japanese gentian, stock, rose, dendrobium and lily petals by particle bombardment. GUS staining spots were observed in Eustoma, chrysanthemum, carnation, Japanese gentian and stock. These results showed that the InMYB1 promoter functions in most dicots. Third, to show the InMYB1 promoter utility in molecular breeding, a MIXTA‐like gene function was suppressed or enhanced under the control of InMYB1 promoter in Arabidopsis. The transgenic plant showed a conspicuous morphological change only in the form of wrinkled petals. Based on these results, the InMYB1 promoter can be used as a petal‐specific promoter in molecular breeding of floricultural crops.     

Constitutive or seed‐specific overexpression of Arabidopsis G‐protein γ subunit 3 (AGG3) results in increased seed and oil production and improved stress tolerance in Camelina sativa

Heterotrimeric G‐proteins consisting of Gα, Gβ and Gγ subunits play an integral role in mediating multiple signalling pathways in plants. A novel, recently identified plant‐specific Gγ protein, AGG3, has been proposed to be an important regulator of organ size and mediator of stress responses in Arabidopsis, whereas its potential homologs in rice are major quantitative trait loci for seed size and panicle branching. To evaluate the role of AGG3 towards seed and oil yield improvement, the gene was overexpressed in Camelina sativa, an oilseed crop of the Brassicaceae family. Analysis of multiple homozygous T4 transgenic Camelina lines showed that constitutive overexpression of AGG3 resulted in faster vegetative as well as reproductive growth accompanied by an increase in photosynthetic efficiency. Moreover, when expressed constitutively or specifically in seed tissue, AGG3 was found to increase seed size, seed mass and seed number per plant by 15%–40%, effectively resulting in significantly higher oil yield per plant. AGG3 overexpressing Camelina plants also exhibited improved stress tolerance. These observations draw a strong link between the roles of AGG3 in regulating two critical yield parameters, seed traits and plant stress responses, and reveal an effective biotechnological tool to dramatically increase yield in agricultural crops.     

Expression of the Grifola frondosa Trehalose Synthase Gene and Improvement of Drought-Tolerance in Sugarcane （Saccharum officinarum L.）

Trehalose Is a nonreduclng dlsaccharlde of glucose that functions as a protectant In the stabilization of blologlcal structures and enhances stress tolerance to abiotic stresses in organisms. We report here the expression of a Grlfola frondosa trehalose synthase （TSase） gene for Improving drought tolerance In sugarcane （Saccharum offlclnarum L.）. The expression of the transgene was under the control of two tandem copies of the CaMV35S promoter and transferred Into sugarcane by Agrobacterium tumefaciens EHA105. The transgenlc plants accumulated high levels of trehalose, up to 8.805-12.863 mg/g fresh weight, whereas It was present at undetectable level in nontransgenlc plants. It has been reported that transgenlc plants transformed with Escherlchla coil TPS （trehalose-6-phosphatesynthase） and/or TPP （trehalose-6-phosphate phosphatase） are severely stunted and have root morphologlc alterations. Interestingly, our transgenlc sugarcane plants had no obvious morphological changes and no growth Inhibition in the field. Trehalose accumulation in 35S-35S：TSase plants resulted In In- creased drought tolerance, as shown by the drought and the drought physiological Indexes, such as the rate of bound water/free water, plasma membrane permeability, malondlaldehyde content, chlorophyll a and b contents, and activity of SOD and POD of the excised leaves. These results suggest that transgenlc plants transformed with the TSase gene can accumulate high levels of trehalose and have enhanced tolerance to drought.