A Case Study from the Overexpression of OsTZF5, Encoding a CCCH Tandem Zinc Finger Protein, in Rice Plants Across Nineteen Yield Trials.

BACKGROUND: Development of transgenic rice overexpressing transcription factors involved in drought response has been previously reported to confer drought tolerance and therefore represents a means of crop improvement. We transformed lowland rice IR64 with OsTZF5, encoding a CCCH-tandem zinc finger protein, under the control of the rice LIP9 stress-inducible promoter and compared the drought response of transgenic lines and nulls to IR64 in successive screenhouse paddy and field trials up to the T6 generation. RESULTS: Compared to the well-watered conditions, the level of drought stress across experiments varied from a minimum of - 25 to - 75 kPa at a soil depth of 30 cm which reduced biomass by 30-55% and grain yield by 1-92%, presenting a range of drought severities. OsTZF5 transgenic lines showed high yield advantage under drought over IR64 in early generations, which was related to shorter time to flowering, lower shoot biomass and higher harvest index. However, the increases in values for yield and related traits in the transgenics became smaller over successive generations despite continued detection of drought-induced transgene expression as conferred by the LIP9 promoter. The decreased advantage of the transgenics over generations tended to coincide with increased levels of homozygosity. Background cleaning of the transgenic lines as well as introgression of the transgene into an IR64 line containing major-effect drought yield QTLs, which were evaluated starting at the BC3F1 and BC2F3 generation, respectively, did not result in consistently increased yield under drought as compared to the respective checks. CONCLUSIONS: Although we cannot conclusively explain the genetic factors behind the loss of yield advantage of the transgenics under drought across generations, our results help in distinguishing among potential drought tolerance mechanisms related to effectiveness of the transgenics, since early flowering and harvest index most closely reflected the levels of yield advantage in the transgenics across generations while reduced biomass did not.

STOP1-regulated SMALL AUXIN UP RNA55 (SAUR55) is involved in proton/malate co-secretion for Al tolerance in Arabidopsis.

Proton (H+) release is linked to aluminum (Al)-enhanced organic acids (OAs) excretion from the roots under Al rhizotoxicity in plants. It is well-reported that the Al-enhanced organic acid excretion mechanism is regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1), a zinc-finger TF that regulates major Al tolerance genes. However, the mechanism of H+ release linked to OAs excretion under Al stress has not been fully elucidated. Recent physiological and molecular-genetic studies have implicated the involvement of SMALL AUXIN UP RNAs (SAURs) in the activation of plasma membrane H+-ATPases for stress responses in plants. We hypothesized that STOP1 is involved in the regulation of Al-responsive SAURs, which may contribute to the co-secretion of protons and malate under Al stress conditions. In our transcriptome analysis of the roots of the stop1 (sensitive to proton rhizotoxicity1) mutant, we found that STOP1 regulates the transcription of one of the SAURs, namely SAUR55. Furthermore, we observed that the expression of SAUR55 was induced by Al and repressed in the STOP1 T-DNA insertion knockout (KO) mutant (STOP1-KO). Through in silico analysis, we identified a functional STOP1-binding site in the promoter of SAUR55. Subsequent in vitro and in vivo studies confirmed that STOP1 directly binds to the promoter of SAUR55. This suggests that STOP1 directly regulates the expression of SAUR55 under Al stress. We next examined proton release in the rhizosphere and malate excretion in the T-DNA insertion KO mutant of SAUR55 (saur55), in conjunction with STOP1-KO. Both saur55 and STOP1-KO suppressed rhizosphere acidification and malate release under Al stress. Additionally, the root growth of saur55 was sensitive to Al-containing media. In contrast, the overexpressed line of SAUR55 enhanced rhizosphere acidification and malate release, leading to increased Al tolerance. These associations with Al tolerance were also observed in natural variations of Arabidopsis. These findings demonstrate that transcriptional regulation of SAUR55 by STOP1 positively regulates H+ excretion via PM H+-ATPase 2 which enhances Al tolerance by malate secretion from the roots of Arabidopsis. The activation of PM H+-ATPase 2 by SAUR55 was suggested to be due to PP2C.D2/D5 inhibition by interaction on the plasma membrane with its phosphatase. Furthermore, RNAi-suppression of NtSTOP1 in tobacco shows suppression of rhizosphere acidification under Al stress, which was associated with the suppression of SAUR55 orthologs, which are inducible by Al in tobacco. It suggests that transcriptional regulation of Al-inducible SAURs by STOP1 plays a critical role in OAs excretion in several plant species as an Al tolerance mechanism.

Virus-Mediated Transient Expression Techniques Enable Functional Genomics Studies and Modulations of Betalain Biosynthesis and Plant Height in Quinoa.

Quinoa (Chenopodium quinoa), native to the Andean region of South America, has been recognized as a potentially important crop in terms of global food and nutrition security since it can thrive in harsh environments and has an excellent nutritional profile. Even though challenges of analyzing the complex and heterogeneous allotetraploid genome of quinoa have recently been overcome, with the whole genome-sequencing of quinoa and the creation of genotyped inbred lines, the lack of technology to analyze gene function in planta is a major limiting factor in quinoa research. Here, we demonstrate that two virus-mediated transient expression techniques, virus-induced gene silencing (VIGS) and virus-mediated overexpression (VOX), can be used in quinoa. We show that apple latent spherical virus (ALSV) can induce gene silencing of quinoa phytoene desaturase (CqPDS1) in a broad range of quinoa inbred lines derived from the northern and southern highland and lowland sub-populations. In addition, we show that ALSV can be used as a VOX vector in roots. Our data also indicate that silencing a quinoa 3,4-dihydroxyphenylalanine 4,5-dioxygenase gene (CqDODA1) or a cytochrome P450 enzyme gene (CqCYP76AD1) inhibits betalain production and that knockdown of a reduced-height gene homolog (CqRHT1) causes an overgrowth phenotype in quinoa. Moreover, we show that ALSV can be transmitted to the progeny of quinoa plants. Thus, our findings enable functional genomics in quinoa, ushering in a new era of quinoa research.

CRISPR/Cas9-targeted mutagenesis of OsERA1 confers enhanced responses to abscisic acid and drought stress and increased primary root growth under nonstressed conditions in rice.

Abscisic acid (ABA) signaling components play an important role in the drought stress response in plants. Arabidopsis thaliana ENHANCED RESPONSE TO ABA1 (ERA1) encodes the ß-subunit of farnesyltransferase and regulates ABA signaling and the dehydration response. Therefore, ERA1 is an important candidate gene for enhancing drought tolerance in numerous crops. However, a rice (Oryza sativa) ERA1 homolog has not been characterized previously. Here, we show that rice osera1 mutant lines, harboring CRISPR/Cas9-induced frameshift mutations, exhibit similar leaf growth as control plants but increased primary root growth. The osera1 mutant lines also display increased sensitivity to ABA and an enhanced response to drought stress through stomatal regulation. These results illustrate that OsERA1 is a negative regulator of primary root growth under nonstressed conditions and also of responses to ABA and drought stress in rice. These findings improve our understanding of the role of ABA signaling in the drought stress response in rice and suggest a strategy to genetically improve rice.

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 Oryza sativa CCCH-tandem zinc finger 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.

Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field.

Drought stress has often caused significant decreases in crop production which could be associated with global warming. Enhancing drought tolerance without a grain yield penalty has been a great challenge in crop improvement. Here, we report the Arabidopsis thaliana galactinol synthase 2 gene (AtGolS2) was able to confer drought tolerance and increase grain yield in two different rice (Oryza sativa) genotypes under dry field conditions. The developed transgenic lines expressing AtGolS2 under the control of the constitutive maize ubiquitin promoter (Ubi:AtGolS2) also had higher levels of galactinol than the non-transgenic control. The increased grain yield of the transgenic rice under drought conditions was related to a higher number of panicles, grain fertility and biomass. Extensive confined field trials using Ubi:AtGolS2 transgenic lines in Curinga, tropical japonica and NERICA4, interspecific hybrid across two different seasons and environments revealed the verified lines have the proven field drought tolerance of the Ubi:AtGolS2 transgenic rice. The amended drought tolerance was associated with higher relative water content of leaves, higher photosynthesis activity, lesser reduction in plant growth and faster recovering ability. Collectively, our results provide strong evidence that AtGolS2 is a useful biotechnological tool to reduce grain yield losses in rice beyond genetic differences under field drought stress.

Virus-induced down-regulation of GmERA1A and GmERA1B genes enhances the stomatal response to abscisic acid and drought resistance in soybean.

Drought is a major threat to global soybean production. The limited transformation potential and polyploid nature of soybean have hindered functional analysis of soybean genes. Previous research has implicated farnesylation in the plant's response to abscisic acid (ABA) and drought tolerance. We therefore used virus-induced gene silencing (VIGS) to evaluate farnesyltransferase genes, GmERA1A and GmERA1B (Glycine max Enhanced Response to ABA1-A and -B), as potential targets for increasing drought resistance in soybean. Apple latent spherical virus (ALSV)-mediated GmERA1-down-regulated soybean leaves displayed an enhanced stomatal response to ABA and reduced water loss and wilting under dehydration conditions, suggesting that GmERA1A and GmERA1B negatively regulate ABA signaling in soybean guard cells. The findings provide evidence that the ALSV-VIGS system, which bypasses the need to generate transgenic plants, is a useful tool for analyzing gene function using only a single down-regulated leaf. Thus, the ALSV-VIGS system could constitute part of a next-generation molecular breeding pipeline to accelerate drought resistance breeding in soybean.

Design of an optimal promoter involved in the heat-induced transcriptional pathway in Arabidopsis, soybean, rice and maize.

Interactions between heat shock (HS) factors (HSFs) and heat shock response elements (HSEs) are important during the heat shock response (HSR) of flora and fauna. Moreover, plant HSFs that are involved in heat stress are also involved in abiotic stresses such as dehydration and cold as well as development, cell differentiation and proliferation. Because the specific combination of HSFs and HSEs involved in plants under heat stress remains unclear, the mechanism of their interaction has not yet been utilized in molecular breeding of plants for climate change. For the study reported herein, we compared the sequences of HS-inducible genes and their promoters in Arabidopsis, soybean, rice and maize and then designed an optimal HS-inducible promoter. Our analyses suggest that, for the four species, the abscisic acid-independent, HSE/HSF-dependent transcriptional pathway plays a major role in HS-inducible gene expression. We found that an 18-bp sequence that includes the HSE has an important role in the HSR, and that those sequences could be classified as representative of monocotyledons or dicotyledons. With the HS-inducible promoter designed based on our bioinformatic predictions, we were able to develop an optimal HS-specific inducible promoter for seedlings or single cells in roots. These findings demonstrate the utility of our HS-specific inducible promoter, which we expect will contribute to molecular breeding efforts and cell-targeted gene expression in specific plant tissues.

Analysis of the cell death-inducing ability of the ethylene response factors in group VIII of the AP2/ERF family.

The ethylene response factor (ERF) family is one of the largest families of plant-specific transcription factors. We have shown previously that the overexpression of the gene for NtERF3, a tobacco transcriptional repressor containing the ERF-associated amphiphilic repression (EAR) motif in the C-terminal region, induces hypersensitive reaction (HR)-like cell death. Many EAR motif-containing ERFs, including NtERF3, are clustered in group VIII of the ERF family. In this study, we aimed at revealing the cell death-inducing ability of group VIII ERFs and the correlation between ERFs and HR. The results showed that many of the EAR motif-containing ERFs classified into subgroup VIII-a of Arabidopsis, rice, and tobacco had cell death-inducing ability in tobacco leaves. Seven AtERFs in subgroup VIII-b did not induce cell death; however, some ERFs in subgroup VIII-b of rice and tobacco showed cell death-inducing ability. An expression analysis of group VIII ERFs in HR-inducing tobacco suggested that the cell death-inducing ability of NtERFs was not necessarily associated with induction of HR. In addition, it was revealed that the EAR motif-containing AtERFs in subgroup II-a also showed cell death-inducing ability. The influence of sequence variation in the EAR motif on the ability to induce cell death is also discussed.

Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells.