Heat-tolerant basmati rice engineered by over-expression of hsp101

Rice is sensitive to high-temperature stress at almost all the stages of its growth and development. Considering the crucial role of heat shock protein 101 (Hsp101) in imparting thermotolerance to cells, we introduced Arabidopsis thaliana hsp101 (Athsp101) cDNA into the Pusa basmati 1 cultivar of rice (Oryza sativa L.) by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the rice genome was demonstrated by Southern, northern and western blot analyses. There appeared no adverse effect of over-expression of the transgene on overall growth and development of transformants. The genetic analysis of tested T₁ lines showed that the transgene segregated in a Mendelian fashion. We compared the survival of T₂ transgenic lines after exposure to different levels of high-temperature stress with the untransformed control plants. The transgenic rice lines showed significantly better growth performance in the recovery phase following the stress. This thermotolerance advantage appeared to be solely due to over-expression of Hsp101 as neither the expression of low-molecular-weight heat shock proteins (HSPs) nor of other members of Clp family proteins was altered in the transgenic rice. The production of high temperature tolerant transgenic rice cultivars would provide a stability advantage under supra-optimal temperature regime thereby improving its overall performance.     

MAP kinases function downstream of HSP90 and upstream of mitochondria in TMV resistance gene N-mediated hypersensitive cell death

Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2(DD), a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2(DD)- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2(DD)- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.     

Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis

Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants

We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.     

Pathogen-induced calmodulin isoforms in basal resistance against bacterial and fungal pathogens in tobacco

Thirteen tobacco calmodulin (CaM) genes fall into three distinct amino acid homology types. Wound-inducible type I isoforms NtCaM1 and 2 were moderately induced by tobacco mosaic virus (TMV)-mediated hypersensitive reaction, and the type III isoform NtCaM13 was highly induced, while the type II isoforms NtCaM3-NtCaM12 showed little response. Type I and III knockdown tobacco lines were generated using inverted repeat sequences from NtCaM1 and 13, respectively, to evaluate the contribution of pathogen-induced calmodulins (CaMs) to disease resistance. After specific reduction of type I and III CaM gene expression was confirmed in both transgenic lines, we analyzed the response to TMV infection, and found that TMV susceptibility was slightly enhanced in type III CaM knockdown lines compared with the control line. Resistance to a compatible strain of the bacterial pathogen Ralstonia solanacearum, and fungal pathogens Rhizoctonia solani and Pythium aphanidermatum was significantly lower in type III but not in type I CaM knockdown plants. Expression of jasmonic acid (JA)- and/or ethylene-inducible basic PR genes was not affected in these lines, suggesting that type III CaM isoforms are probably involved in basal defense against necrotrophic pathogens in a manner that is independent of JA and ethylene signaling.     

Cloning,expression analysis and In silico characterization of HSP101: a potential player conferring heat stress in Aegilops speltoides (Tausch) Gren

Heat shock protein (HSP101) function as molecular chaperones and confer thermotolerance to plants. In the present investigation, identification, comprehensive expression analysis, phylogeny and protein modelling of HSP101 gene has been done in Aegilops speltoides accession Pau3583. In the present study, we cloned and in silico characterized a HSP101C gene designated as AsHSP101C-Pau3583. AsHSP101C-Pau3583 is 4180 bp long with seven exons and six introns and encoded a polypeptide of 910 amino acids predicted by FGENESH. We have identified 58 SNPs between the AsHSP101C-Pau3583 and reference gene sequence extracted from Ae. speltoides TGAC assembly. Real-time RT-PCR analysis of expression levels of HSP101 gene in two wheat genotypes under heat stress revealed that gene namely HSP101C was up-regulated in Aegilops speltoides acc. Pau3583 by > fourfold in comparison to Triticum aestivum cv. PBW343 under heat stress signifies that it plays a role in conferring heat tolerance. Sequence comparison and phylogenetic analysis of AsHSP101C-Pau3583 with seven wheat homologs Triticum aestivum, Aegilops speltoides (TGAC), Triticum durum cv Cappelli, Triticum durum cv Strongfield, Triticum monococcum, Aegilops tauschii and Triticum urartu showed significant similarities with highly conserved coding regions and functional domains (AAA, AAA + 2, ClpB domains), suggesting the conserved function of HSP101C in different species. The illustration of the protein models of HSP101C in homologs provided information for the ATP-binding motifs within the nucleotide binding domains (NBD), specific for the chaperone activity. These findings are important and identified SNPs could be used for designing markers for ensuring the transfer of AsHSP101C-Pau3583 gene into hexaploid wheat and its role in heat tolerance.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01005-2.     

Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit translation is regulated in a small subunit-independent manner in the expanded leaves of tobacco

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is composed of small subunits (SSs) encoded by rbcS on the nuclear genome and large subunits (LSs) encoded by rbcL on the chloroplast genome, and it is localized in the chloroplast stroma. Constitutive knockdown of the rbcS gene reportedly causes a reduction in LS quantity and the level of translation in tobacco and the unicellular green alga Chlamydomonas. Constitutively knockdown of the rbcS gene also causes a reduction in photosynthesis, which influences the expression of photosynthetic genes, including the rbcL gene. Here, to investigate the influence of the knockdown of the rbcS gene on the expression of the rbcL gene under normal photosynthetic conditions, we generated transgenic tobacco plants in which the amount of endogenous rbcS mRNA can be reduced by inducible expression of antisense rbcS mRNA with dexamethasone (DEX) treatment at later stages of growth. In already expanded leaves, after DEX treatment, the level of photosynthesis, RuBisCO quantity and the chloroplast ultrastructure were normal, but the amount of rbcS mRNA was reduced. An in vivo pulse labeling experiment and polysome analysis showed that LSs were translated at the same rate as in wild-type leaves. On the other hand, in newly emerging leaves, the rbcS mRNA quantity, the level of photosynthesis and the quantity of RuBisCO were reduced, and chloroplasts failed to develop. In these leaves, the level of LS translation was inhibited, as previously described. These results suggest that LS translation is regulated in an SS-independent manner in expanded leaves under normal photosynthetic conditions.     

Transgenic tobacco plants expressingBoRS1 gene fromBrassica oleracea var.acephala show enhanced tolerance to water stress

Water stress is by far the leading environmental stress limiting crop yields worldwide. Genetic engineering techniques hold great promise for developing crop cultivars with high tolerance to water stress. In this study, theBrassica oleracea var.acephala BoRS1 gene was transferred into tobacco throughAgrobacterium- mediated leaf disc transformation. The transgenic status and transgene expression of the transgenic plants was confirmed by polymerase chain reaction (PCR) analysis, Southern hybridization and semi-quantitative one step RT-PCR analysis respectively. Subsequently, the growth status under water stress, and physiological responses to water stress of transgenic tobacco were studied. The results showed that the transgenic plants exhibited better growth status under water stress condition compared to the untransformed control plants. In physiological assessment of water tolerance, transgenic plants showed more dry matter accumulation and maintained significantly higher levels of leaf chlorophyll content along with increasing levels of water stress than the untransformed control plants. This study shows thatBoRS1 is a candidate gene in the engineering of crops for enhanced water stress tolerance.     

Production of transgenic male sterile tobacco plants with the cDNA encoding a ribosome inactivating protein in Dianthus sinensis L.

The ribosome inactivating protein (RIP) gene from D. sinensis was used as a cytotoxin gene to induce male sterility in tobacco plants. The TA29 promoter, obtained by PCR amplification from tobacco, was fused to the RIP cDNA, and the chimaeric molecule was then introduced into tobacco plants by Agrobacterium-mediated transformation. Out of twenty-one independent transformants, twenty transgenic tobacco plants exhibited male sterility. Southern blot analysis revealed that four of the transgenic plants contained a single copy of the RIP gene, while the rest of the transgenic tobacco plants had two to four copies of the gene. The transgenic male sterile plants set seeds normally when pollinated with pollens from untransformed control plants, indicating that the RIP gene does not affect the pistil development. Furthermore, the seed yield of the transgenic plant was similar to that of the untransformed, self-pollinated control plant. A light microscopic observation of anther cross sections clearly showed that the tapetal tissue of the anther was selectively and completely destroyed causing male sterility. This study suggests that the RIP gene can be used as a cytotoxin gene for induction of male sterility in the plant.     

Transgenic Nicotiana tabacum and Arabidopsis thaliana plants overexpressing allene oxide synthase

 Allene oxide synthase (AOS), encoded by a single gene in Arabidopsis thaliana (L.) Heynh., catalyzes the first step specific to the octadecanoid pathway. Enzyme activity is very low in control plants, but is upregulated by wounding, octadecanoids, ethylene, salicylate and coronatine (D. Laudert and E.W. Weiler, 1998, Plant J 15: 675–684). In order to study the consequences of constitutive expression of AOS on the level of jasmonates, a complete cDNA encoding the enzyme from A. thaliana was constitutively expressed in both  A. thaliana and tobacco (Nicotiana tabacum L.). Overexpression of AOS did not alter the basal level of jasmonic acid; thus, output of the jasmonate pathway in the unchallenged plant appears to be strictly limited by substrate availability. In wounded plants overexpressing AOS, peak jasmonate levels were 2- to 3-fold higher compared to untransformed plants. More importantly, the transgenic plants reached the maximum jasmonate levels significantly earlier than wounded untransformed control plants. These findings suggest that overexpression of AOS might be a way of controlling defense dynamics in higher plants. Received: 10 February 2000 / Accepted: 11 March 2000     

Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene

GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the Smirnoff-Wheeler's pathway for the biosynthesis of ascorbic acid (AsA) in plants. We have reported recently that the expression of the acerola (Malpighia glabra) GMP gene, designated MgGMP, correlates with the AsA content of the plant. The acerola plant has very high levels of AsA relative to better studied model plants such as Arabidopsis. Here we found that the GMP mRNA levels in acerola are higher than those from Arabidopsis and tomato. Also, the transient expression of the uidA reporter gene in the protoplasts of Nicotiana tabacum cultures showed the MgGMP gene promoter to have higher activity than the cauliflower mosaic virus 35S and Arabidopsis GMP promoters. The AsA content of transgenic tobacco plants expressing the MgGMP gene including its promoter was about 2-fold higher than that of the wild type.     

Expression of the algal cytochrome c6 gene in Arabidopsis enhances photosynthesis and growth

Photosynthetic plants convert light energy into ATP and NADPH in photosynthetic electron transfer and photophosphorylation, and synthesize mainly carbohydrates in the Calvin-Benson cycle. Here we report the enhancement of photosynthesis and growth of plants by introducing the gene of an algal cytochrome c6, which has been evolutionarily eliminated from higher plant chloroplasts, into the model plant Arabidopsis thaliana. At 60 d after planting, the plant height, leaf length and root length of the transformants were 1.3-, 1.1- and 1.3-fold those in the wild-type plants, respectively. At the same time, in the transgenic plants, the amounts of chlorophyll, protein, ATP, NADPH and starch were 1.2-, 1.1-, 1.9-, 1.4- and 1.2-fold those in the wild-type plants, respectively. The CO2 assimilation capacity of the transgenic plants was 1.3-fold that of the wild type. Moreover, in transgenic Arabidopsis expressing algal cytochrome c6, the 1-qP, which reflects the reduced state of the plastoquinone pool, is 30% decreased compared with the wild type. These results show that the electron transfer of photosynthesis of Arabidopsis would be accelerated by the expression of algal cytochrome c6. Our results demonstrate that the growth and photosynthesis of Arabidopsis plants could be enhanced by the expression of the algal cytochrome c6 gene.     

Maize HSP101 plays important roles in both induced and basal thermotolerance and primary root growth

HSP101 belongs to the ClpB protein subfamily whose members promote the renaturation of protein aggregates and are essential for the induction of thermotolerance. We found that maize HSP101 accumulated in mature kernels in the absence of heat stress. At optimal temperatures, HSP101 disappeared within the first 3 days after imbibition, although its levels increased in response to heat shock. In embryonic cells, HSP101 concentrated in the nucleus and in some nucleoli. Hsp101 maps near the umc132 and npi280 markers on chromosome 6. Five maize hsp101-m-::Mu1 alleles were isolated. Mutants were null for HSP101 and defective in both induced and basal thermotolerance. Moreover, during the first 3 days after imbibition, primary roots grew faster in the mutants at optimal temperature. Thus, HSP101 is a nucleus-localized protein that, in addition to its role in thermotolerance, negatively influences the growth rate of the primary root. HSP101 is dispensable for proper embryo and whole plant development in the absence of heat stress.     

Four members of the HSP101 gene family are differently regulated in Triticum durum Desf

Heat shock proteins play an essential role in preventing deleterious effects of high temperatures. In many plants, HSP101 has a central role in heat stress survival. We report the isolation and characterization of four cDNAs corresponding to different members of the durum wheat HSP101 gene family. Expression analysis revealed differences in their induction. Accordingly, durum wheat HSP101 genes are differently regulated, therefore having distinct roles in stress response and thermotolerance acquisition. These findings are important for further dissection of the molecular mechanisms underlying the stress response and for understanding the functions of the HSP101 family members. This information could be important for the exploitation of specific alleles in marker assisted selection for abiotic stress resistance.