Cytonuclear interactions modulate the plasticity of photosynthetic rhythmicity and growth in wild barley.

In plants, the contribution of the plasmotype (mitochondria and chloroplast) in controlling the circadian clock plasticity and possible consequences on cytonuclear genetic makeup have yet to be fully elucidated. A genome-wide association study in the wild barley (Hordeum vulgare ssp. spontaneum) B1K collection identified overlap with our previously mapped DRIVERS OF CLOCKS (DOCs) loci in wild-cultivated interspecific population. Moreover, we identified non-random segregation and epistatic interactions between nuclear DOCs loci and the chloroplastic RpoC1 gene, indicating an adaptive value for specific cytonuclear gene combinations. Furthermore, we show that DOC1.1, which harbours the candidate SIGMA FACTOR-B (SIG-B) gene, is linked with the differential expression of SIG-B and CCA1 genes and contributes to the circadian gating response to heat. High-resolution temporal growth and photosynthesis measurements of B1K also link the DOCs loci to differential growth, Chl content and quantum yield. To validate the involvement of the Plastid encoded polymerase (PEP) complex, we over-expressed the two barley chloroplastic RpoC1 alleles in Arabidopsis and identified significant differential plasticity under elevated temperatures. Finally, enhanced clock plasticity of de novo ENU (N-Ethyl-N-nitrosourea) -induced barley rpoB1 mutant further implicates the PEP complex as a key player in regulating the circadian clock output. Overall, this study highlights the contribution of specific cytonuclear interaction between rpoC1 (PEP gene) and SIG-B with distinct circadian timing regulation under heat, and their pleiotropic effects on growth implicate an adaptive value.

Crops under past diversification and ongoing climate change: more than just selection of nuclear genes for flowering.

Diversification and breeding following domestication and under current climate change across the globe are the two most significant evolutionary events experienced by major crops. Diversification of crops from their wild ancestors has favored dramatic changes in the sensitivity of the plants to the environment, particularly significantly in transducing light inputs to the circadian clock, which has allowed the growth of major crops in the relatively short growing season experienced in the Northern Hemisphere. Historically, mutants and the mapping of quantitative trait loci (QTL) have facilitated the identification and the cloning of genes that underlie major changes of the clock and the regulation of flowering. Recent studies have suggested that the thermal plasticity of the circadian clock output, and not just the core genes that follow temperature compensation, has also been under selection during diversification and breeding. Wild alleles that accelerate output rhythmicity could be beneficial for crop resilience. Furthermore, wild alleles with beneficial and flowering-independent effects under stress indicate their possible role in maintaining a balanced source-sink relationship, thereby allowing productivity under climatic change. Because the chloroplast genome also regulates the plasticity of the clock output, mapping populations including cytonuclear interactions should be utilized within an integrated field and clock phenomics framework. In this review, we highlight the need to integrate physiological and developmental approaches (physio-devo) to gain a better understanding when re-domesticating wild gene alleles into modern cultivars to increase their robustness under abiotic heat and drought stresses.

Arabidopsis plants overexpressing additional copies of heat shock protein Hsp101 showed high heat tolerance and endo-gene silencing.

Hsp101 chaperone is vital for survival of plants under heat stress. We generated transgenic Arabidopsis thaliana (Arabidopsis) lines with extra copies of Hsp101 gene using diverse approaches. Arabidopsis plants transformed with rice Hsp101 cDNA driven by Arabidopsis Hsp101 promoter (IN lines) showed high heat tolerance while the plants transformed with rice Hsp101 cDNA driven by CaMV35S promoter (C lines) were like wild type plants in heat stress response. Transformation of Col-0 plants with 4633 bp Hsp101 genomic fragment (GF lines) from A. thaliana containing both its coding and the regulatory sequence resulted in mostly over-expressor (OX) lines and a few under-expressor (UX) lines of Hsp101. OX lines showed enhanced heat tolerance while the UX lines were overly heat sensitive. In UX lines, silencing of not only Hsp101 endo-gene was noted but also transcript of choline kinase (CK2) was silenced. Previous work established that in Arabidopsis, CK2 and Hsp101 are convergent gene pairs sharing a bidirectional promoter. The elevated AtHsp101 protein amount in most GF and IN lines was accompanied by lowered CK2 transcript levels under HS. We observed increased methylation of the promoter and gene sequence region in UX lines; however, methylation was lacking in OX lines.

Cpn60ß4 protein regulates growth and developmental cycling and has bearing on flowering time in Arabidopsis thaliana plants.

Chloroplastic Cpn60 proteins are type I chaperonins comprising of Cpn60α and Cpn60ß subunits. Arabidopsis genome contains six entries in Cpn60 family, out of which two are for Cpn60α subunit and four for Cpn60ß subunit. We noted that the cpn60ß4 knockout mutant plants (T-DNA insertion salk_064887 line) differed from the wild type Col-0 plants in the developmental programming. cpn60ß4 mutant plants showed early seed germination. Radical emergence, hypocotyl emergence and cotyledons opening were faster in cpn60ß4 mutant plants than WT. Importantly, cpn60ß4 mutant plants showed early-flowering phenotype. The number of flowers and siliques as well as weight of the seeds were higher in cpn60ß4 mutant plants as compared to Col-0 plants. These effects were reverted to wild type like growth and developmental patterns when genomic fragment of Arabidopsis encompassing Cpn60ß4 gene was complemented in the mutant background. The overexpression of Cpn60ß4 gene using CaMV35 promoter in wild type background (OE-Cpn60ß4) delayed the floral transition as against wild type plants. The plastid division were affected in cpn60ß4 mutant plants compared to Col-0. The results of this study suggest that Cpn60ß4 plays important role(s) in chloroplast development and is a key factor in plant growth, development and flowering in Arabidopsis.

Characterization of 5'UTR of rice ClpB-C/Hsp100 gene: evidence of its involvement in post-transcriptional regulation.

Rice (Oryza sativa) ClpB-C (OsClpB-C) protein is expressed upon heat stress in vegetative tissues and constitutively in seeds. We produced stably transformed Arabidopsis plants carrying ß-glucuronidase (Gus) reporter gene downstream to 1-kb OsClpB-C promoter (1kbPro plants). In the 1kbPro plants, expression of Gus transcript and protein followed the expression pattern of OsClpB-C gene in rice plants, i.e., heat induced in vegetative tissues and constitutive in seeds. Next, we produced transgenic Arabidopsis plants containing Gus downstream to 862-bp fragment of OsClpB-C promoter [lacking 138 nucleotides from 3' end of the 5'untranslated region (5'UTR); ∆UTR plants). In ∆UTR plants, Gus transcript was expressed in heat-inducible manner, but strikingly, Gus protein levels were negligible after heat treatment. However, Gus protein was expressed in ∆UTR seedlings at levels comparable to 1kbPro seedlings when recovery treatment of 22 °C/2 h was given post heat stress (38 °C/15 min). This suggests that 5'UTR of OsClpB-C gene is involved in its post-transcriptional regulation and is an obligate requirement for protein expression during persistent heat stress. Furthermore, the Gus transcript levels were higher in the polysomal RNA fraction in heat-stressed seedlings of 1kbPro plants as compared to ∆UTR plants, indicating that 5'UTR aids in assembly of ribosomes onto the Gus transcript during heat stress. Unlike the case of seedlings, Gus protein was formed constitutively in ∆UTR seeds at levels comparable to 1kbPro seeds. Hence, the function of 5'UTR of OsClpB-C is dispensable for expression in seeds.

Constitutive over-expression of rice chymotrypsin protease inhibitor gene OCPI2 results in enhanced growth, salinity and osmotic stress tolerance of the transgenic Arabidopsis plants.

Protease inhibitors are involved primarily in defense against pathogens. In recent years, these proteins have also been widely implicated in response of plants to diverse abiotic stresses. Rice chymotrypsin protease inhibitor gene OCPI2 is highly induced under salt and osmotic stresses. The construct containing the complete coding sequence of OCPI2 cloned downstream to CaMV35S promoter was transformed in Arabidopsis and single copy, homozygous transgenic lines were produced. The transgenic plants exhibited significantly enhanced tolerance to NaCl, PEG and mannitol stress as compared to wild type plants. Importantly, the vegetative and reproductive growth of transgenic plants under unstressed, control conditions was also enhanced: transgenic plants were more vigorous than wild type, resulting into higher yield in terms of silique number. The RWC values and membrane stability index of transgenic in comparison to wild type plants was higher. Higher proline content was observed in the AtOCPI2 lines, which was associated with higher transcript expression of pyrroline-5-carboxylate synthase and lowered levels of proline dehydrogenase genes. The chymotrypsin protease activities were lower in the transgenic as against wild type plants, under both unstressed, control as well as stressed conditions. It thus appears that rice chymotrypsin protease inhibitor gene OCPI2 is a useful candidate gene for genetic improvement of plants against salt and osmotic stress.