Improving tobacco freezing tolerance by co-transfer of stress-inducible <Emphasis Type="Italic">CbCBF</Emphasis> and <Emphasis Type="Italic">CbICE53</Emphasis> genes

Cold stress is one of the major limitations to crop productivity worldwide. We investigated the effects of multiple gene expression from cold tolerant Capsella bursa-pastoris in transgenic tobacco (Nicotiana tabaccum) plants. We combined CblCE53 and CbCBF into a reconstruct vector by isocaudomers. Plant overexpression of CbICE53 under the stress inducible CbCOR15b promoter and CbCBF under a constitutive promoter showed increased tolerance to both chilling and freezing temperatures in comparison to wild-type plants, according to the electrolyte leakage and relative water content. The expressions of endogenous cold-responsive genes in transgenic tobacco (NtDREB1, NtDREB3, NtERD10a and NtERD10b) were obviously upregulated under normal and low temperature conditions. These results suggest that the CbICE53 + CbCBF transgenic plants showed a much greater cold tolerance as well as no dwarfism and delayed flowering. Thus they can be considered as a potential candidate for transgenic engineering for cold tolerant tobacco.     

<Emphasis Type="Italic">ZmFKBP20-1</Emphasis> improves the drought and salt tolerance of transformed Arabidopsis

FK506-binding proteins (FKBPs), which belong to the peptidyl-prolyl cis/trans isomerase superfamily, are involved in plant response to abiotic stresses. A number of FKBP family genes have been isolated in plants, but little has been reported of FKBP genes in maize. In this study, a drought-induced FKBP gene, ZmFKBP20-1, was isolated from maize and was characterized for its role in stress responses using gene expression, protein subcellular localization, transformation in Arabidopsis, expression patterns of the stress-responsive genes, and physiological parameter analysis. During drought and salt stresses, ZmFKBP20-1 transgenic Arabidopsis plants exhibited enhanced tolerance, which was concomitant with the altered expression of stress/ABA-responsive genes, such as COR15a, COR47, ERD10, RD22, KIN1, ABI1, and ABI2. The resistance characteristics of ZmFKBP20-1 overexpression were associated with a significant increase in survival rate. These results suggested that ZmFKBP20-1 plays a positive role in drought and salt stress responses in Arabidopsis and provided new insights into the mechanisms of FKBP in response to abiotic stresses in plants.     

<Emphasis Type="Italic">Platycladus orientalis</Emphasis> PoKub3 is involved in abiotic stress responses in transgenic arabidopsis

Ku70-binding proteins associate with Ku70 and their expression levels can affect DSB repair efficiency via the DNA-PK-dependent repair pathway. However, how Ku70-binding proteins in plants exert a regulatory function under abiotic stress is poorly understood. Here, we cloned and characterized a PoKub3 gene from 500-year-old Platycladus orientalis. With increasing age, PoKub3 expression in P. orientalis increased gradually. The PoKub3 expression levels in leaves were upregulated under salt, heat, UV-C and abscisic acid treatments according to qRT-PCR. Moreover, PoKub3 overexpression in Arabidopsis thaliana improved tolerance to salt and drought stress compared with wild-type (WT) and vector control (VC) plants. High RAB18 and DREB2A expression and low JAZ1 and ABI2 expression provided strong evidence that salt tolerance was enhanced in the overexpression plants. Similarly, high RAB18 and DREB2A expression, accompanied by low JAZ1 and LOX1 expression and high DREB1A, CPK10, GSTF6 and APX1 expression, suggested the drought tolerance mechanism was associated with the abscisic acid pathway. In addition, lower malondialdehyde content, electrolyte leakage and stomatal conductance, and higher soluble sugar and relative water contents in PoKub3 overexpression lines than in WT and VC plants demonstrated its role in salt and drought tolerance. Together, these findings show that PoKub3 positively regulates salt and drought tolerance by regulating stress-related genes.     

Isolation and characterization of two distinct Class II <Emphasis Type="Italic">PR4</Emphasis> genes from the oriental lily hybrid Sorbonne

Pathogenesis-related (PR) proteins are generally involved in the defense of plants and are important contributors in the disease resistance of plants. Among the 17 PRs that are currently recognized, the PR4 family of proteins is divided into two classes and features a conserved barwin domain. In this study, we isolated two Class II PR4s from the oriental hybrid lily cultivar Sorbonne using the rapid amplification of the cDNA ends (RACE) method, and designated these two PR4s LhSorPR4a and LhSorPR4b. LhSorPR4a and LhSorPR4b were 627 and 617 bp in length, respectively, and encoded two corresponding PR4s of 141 and 143 amino acids. These deciphered LhSorPR4a and LhSorPR4b protein sequences shared a sequence similarity of 90.7%, but their theoretical isoelectric points were distinctively different (7.74 and 4.08, respectively). The three-dimensional structures of LhSorPR4a and LhSorPR4b predicted by homology modeling showed high similarity to their corresponding papaya barwin-like protein template. Analysis of expression by qPCR revealed that both LhSorPR4a and LhSorPR4b were responsive to methyl jasmonate and ethephon treatments. The LhSorPR4b expression was also significantly induced by sodium salicylate (SS); however, LhSorPR4a was unresponsive to the SS treatment. Both LhSorPR4a and LhSorPR4b were expressed in Escherichia coli (E. coli) and successfully purified. The PR4s characterized in this study (LhSorPR4a and LhSorPR4b) are the first two PR4 family genes isolated from the Lilium genus, and they could therefore play an important role in lily disease resistance.     

Identification and Functional Analysis of Two Cotton Orthologs of <Emphasis Type="Italic">MAX2</Emphasis> Which Control Shoot Lateral Branching

Cotton (Gossypium spp.), as the most important fiber and oilseed crop in the world, is extremely important for the industry. However, due to its indeterminate growth habit and complex branching system, massive labor costs are needed for shoot apex removal and branch pruning during cotton production. Therefore, it is very important to explore branch-controlling genes and genetically modify the branch architecture of cotton. Strigolactones (SLs) are a novel class of plant hormone that inhibit the outgrowth of lateral branches. To elucidate the role of SLs in branch development of cotton, we cloned and characterized GhMAX2a and GhMAX2b from tetraploid upland cotton (Gossypium hirsutum), the orthologs of Arabidopsis MAX2, rice D3, and petunia RMS4. GhMAX2a/2b was ubiquitously expressed in all tested tissues of cotton, with relatively higher expression levels in leaves and lateral buds. Subcellular localization assay showed that the GhMAX2-GFP fusion protein localized to the nucleus. Both GhMAX2a and GhMAX2b can fully rescue the dwarfed and highly branched phenotypes of the Arabidopsis max2-1 mutant, indicating that GhMAX2s have conserved functions with that of AtMAX2. The cotton GhMAX2b interacted with Arabidopsis Skp1-like 1 (ASK1) proteins in vitro which was further confirmed in the Arabidopsis protoplasts using the co-immunoprecipitation assay, indicating that GhMAX2b probably functions through forming an SCF E3 complex with Skp and other proteins in the Arabidopsis. These results suggest that the cotton GhMAX2s encode functional MAX2 that can inhibit the shoot lateral branching. Further functional analysis of GhMAX2s in determining cotton branch architecture and yield is underway.     

Arabidopsis PRC1 core component AtRING1 regulates stem cell-determining carpel development mainly through repression of class I <Emphasis Type="Italic">KNOX</Emphasis> genes

Background

Polycomb repressive complex 2 (PRC2)-catalyzed H3K27me3 marks are tightly associated with the WUS-AG negative feedback loop to terminate floral stem cell fate to promote carpel development, but the roles of Polycomb repressive complex 1 (PRC1) in this event remain largely uncharacterized.

Results

Here we show conspicuous variability in the morphology and number of carpels among individual flowers in the absence of the PRC1 core components AtRING1a and AtRING1b, which contrasts with the wild-type floral meristem consumed by uniform carpel production in Arabidopsis thaliana. Promoter-driven GUS reporter analysis showed that AtRING1a and AtRING1b display a largely similar expression pattern, except in the case of the exclusively maternal-preferred expression of AtRING1b, but not AtRING1a, in the endosperm. Indeterminate carpel development in the atring1a;atring1b double mutant is due to replum/ovule-to-carpel conversion in association with ectopic expression of class I KNOX (KNOX-I) genes. Moreover, AtRING1a and AtRING1b also play a critical role in ovule development, mainly through promoting the degeneration of non-functional megaspores and proper integument formation. Genetic interaction analysis indicates that the AtRING1a/b-regulated KNOX-I pathway acts largely in a complementary manner with the WUS-AG pathway in controlling floral stem cell maintenance and proper carpel development.

Conclusions

Our study uncovers a novel mechanistic pathway through which AtRING1a and AtRING1b repress KNOX-I expression to terminate floral stem cell activities and establish carpel cell fate identities.

     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

High irradiance sensitive phenotype of <Emphasis Type="Italic">Arabidopsis hit2/xpo1a</Emphasis> mutant is caused in part by nuclear confinement of AtHsfA4a

In Arabidopsis, EXPORTIN1A (HIT2/XPO1A) and EXPORTIN1B (XPO1B) mediate the translocation of nuclear export sequence (NES)-bearing proteins from nucleus to cytoplasm. However, a mutation in HIT2/XPO1A but not in XPO1B induces sensitivity to high irradiance (HI). Arabidopsis thaliana heat stress elements A4a and A5 (AtHsfA4a and AtHsfA5) are involved in plant responses to HI and possess NESs; therefore, their nucleo-cytoplasmic partitioning was analyzed. In wild-type and xpo1b mutant cells, AtHsfA4a normally remained in the cytoplasm but became concentrated in the nucleus following exposure to HI, whereas AtHsfA5 was constitutively distributed in both cytoplasm and nucleus. However, in hit2/xpo1a mutant, AtHsfA4a and AtHsfA5 were always confined to the nucleus, regardless of the irradiance. Although AtHsfA4a can enhance the ability of plants to scavenge H₂O₂, and AtHsfA5 is a repressor of AtHsfA4a, athsfa5 but not athsfa4a mutant plants exhibited HI sensitivity. Additionally, athsfa4a plants expressing AtHsfA4aΔNES were sensitive to HI, but athsfa5 plants expressing AtHsfA5ΔNES were not. Meanwhile, hit2/athsfa4a double mutant was more tolerant to HI than hit2. These results indicate that both AtHsfA4a and AtHsfA5 were HIT2/XPO1A-specific substrates. Long-term accumulation of AtHsfA4a contributed to the hit2 HI-sensitive phenotype independent of the scavenging ability of H₂O₂, and the presence of AtHsfA5 could mitigate this adverse effect.     

Analysis of an N-terminal deletion in subunit <Emphasis Type="Italic">a</Emphasis> of the <Emphasis Type="Italic">Escherichia coli</Emphasis> ATP synthase

Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6–20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.     

Identification and characterization of <Emphasis Type="Italic">Rht25</Emphasis>, a locus on chromosome arm 6AS affecting wheat plant height,heading time,and spike development

Key message

This study identified Rht25, a new plant height locus on wheat chromosome arm 6AS, and characterized its pleiotropic effects on important agronomic traits.

Abstract

Understanding genes regulating wheat plant height is important to optimize harvest index and maximize grain yield. In modern wheat varieties grown under high-input conditions, the gibberellin-insensitive semi-dwarfing alleles Rht-B1b and Rht-D1b have been used extensively to confer lodging tolerance and improve harvest index. However, negative pleiotropic effects of these alleles (e.g., poor seedling emergence and reduced biomass) can cause yield losses in hot and dry environments. As part of current efforts to diversify the dwarfing alleles used in wheat breeding, we identified a quantitative trait locus (QHt.ucw-6AS) affecting plant height in the proximal region of chromosome arm 6AS (<?0.4 cM from the centromere). Using a large segregating population (~?2800 gametes) and extensive progeny tests (70–93 plants per recombinant family), we mapped QHt.ucw-6AS as a Mendelian locus to a 0.2 cM interval (144.0–148.3 Mb, IWGSC Ref Seq v1.0) and show that it is different from Rht18. QHt.ucw-6AS is officially designated as Rht25, with Rht25a representing the height-increasing allele and Rht25b the dwarfing allele. The average dwarfing effect of Rht25b was found to be approximately half of the effect observed for Rht-B1b and Rht-D1b, and the effect is greater in the presence of the height-increasing Rht-B1a and Rht-D1a alleles than in the presence of the dwarfing alleles. Rht25b is gibberellin-sensitive and shows significant pleiotropic effects on coleoptile length, heading date, spike length, spikelet number, spikelet density, and grain weight. Rht25 represents a new alternative dwarfing locus that should be evaluated for its potential to improve wheat yield in different environments.