Silencing PHOSPHOENOLPYRUVATE CARBOXYLASE1 in the Obligate Crassulacean Acid Metabolism Species Kalanchoë laxiflora causes Reversion to C3-like Metabolism and Amplifies Rhythmicity in a Subset of Core Circadian Clock Genes

ABSTRACT Unlike C 3 plants, Crassulacean acid metabolism (CAM) plants fix CO 2 in the dark using phosphoenolpyruvate carboxylase (PPC; EC 4.1.1.31). PPC combines PEP with CO 2 (as HCO 3 − ), forming oxaloacetate that is rapidly converted to malate, leading to vacuolar malic acid accumulation that peaks phased to dawn. In the light period, malate decarboxylation concentrates CO 2 around RuBisCO for secondary fixation. CAM mutants lacking PPC have not been described. Here, RNAi was employed to silence CAM isogene PPC1 in Kalanchoë laxiflora . Line rPPC1-B lacked PPC1 transcripts, PPC activity, dark period CO 2 fixation, and nocturnal malate accumulation. Light period stomatal closure was also perturbed, and the plants displayed reduced but detectable dark period stomatal conductance, and arrhythmia of the CAM CO 2 fixation circadian rhythm under constant light and temperature (LL) free-running conditions. By contrast, the rhythm of delayed fluorescence was enhanced in plants lacking PPC1 . Furthermore, a subset of gene transcripts within the central circadian oscillator were up-regulated and oscillated robustly. The regulation guard cell genes involved controlling stomatal movements was also altered in rPPC1-B . This provided direct evidence that altered regulatory patterns of key guard cell signaling genes are linked with the characteristic inverse pattern of stomatal opening and closing during CAM

A bipartite transcription factor module controlling expression in the bundle sheath of Arabidopsis thaliana.

C4 photosynthesis evolved repeatedly from the ancestral C3 state, improving photosynthetic efficiency by ~50%. In most C4 lineages, photosynthesis is compartmented between mesophyll and bundle sheath cells, but how gene expression is restricted to these cell types is poorly understood. Using the C3 model Arabidopsis thaliana, we identified cis-elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressed MYB76 gene, we identified a region necessary and sufficient for expression containing two cis-elements associated with the MYC and MYB families of transcription factors. MYB76 expression is reduced in mutant alleles for these transcription factors. Moreover, downregulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with the evolution of C4 photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.ERC BBSR

The starch-deficient plastidic PHOSPHOGLUCOMUTASE mutant of the constitutive crassulacean acid metabolism (CAM) species Kalanchoë fedtschenkoi impacts diel regulation and timing of stomatal CO2 responsiveness.

Background and aimsCrassulacean acid metabolism (CAM) is a specialised type of photosynthesis characterised by a diel pattern of stomatal opening at night and closure during the day, which increases water-use efficiency. Starch degradation is a key regulator of CAM, providing phosphoenolpyruvate as substrate in the mesophyll for nocturnal assimilation of CO2. Growing recognition of a key role for starch degradation in C3 photosynthesis guard cells for mediating day-time stomatal opening presents the possibility that starch degradation might also impact CAM by regulating the provision of energy and osmolytes to increase guard cell turgor and drive stomatal opening at night. This study tested the hypothesis that the timing of diel starch turnover in CAM guard cells has been re-programmed during evolution to enable nocturnal stomatal opening and day-time closure.MethodsBiochemical and genetic characterisation of wild type and starch-deficient RNAi lines of Kalanchoë fedtschenkoi with reduced activity of plastidic phosphoglucomutase (PGM) constituted a preliminary approach for the understanding of starch metabolism and its implications for stomatal regulation in CAM plants.Key resultsStarch deficiency reduced nocturnal net CO2 uptake, but had negligible impact on nocturnal stomatal opening. In contrast, day-time stomatal closure was reduced in magnitude and duration in the starch-deficient rPGM RNAi lines, and their stomata were unable to remain closed in response to elevated concentrations of atmospheric CO2 administered during the day. Curtailed day-time stomatal closure was linked to higher soluble sugar contents in the epidermis and mesophyll.ConclusionsNocturnal stomatal opening is not reliant upon starch degradation, but starch biosynthesis is an important sink for carbohydrates, ensuring day-time stomatal closure in this CAM species

Both male and female meiosis contribute to non‐Mendelian inheritance of parental chromosomes in interspecific plant hybrids (Lolium x Festuca)

Some interspecific plant hybrids show unequal transmission of chromosomes from parental genomes to the successive generations. It has been suggested that this is due to a differential behavior of parental chromosomes during meiosis. However, underlying mechanism is unknown. We analyzed chromosome composition of the F2 generation of Festuca × Lolium hybrids and reciprocal backcrosses to elucidate effects of male and female meiosis on the shift in parental genome composition. We studied male meiosis, including the attachment of chromosomes to the karyokinetic spindle and gene expression profiling of the kinetochore genes. We found that Lolium and Festuca homoeologues were transmitted differently to the F2 generation. Female meiosis led to the replacement of Festuca chromosomes by their Lolium counterparts. In male meiosis, Festuca univalents were attached less frequently to microtubules than Lolium univalents, lagged in divisions and formed micronuclei, which were subsequently eliminated. Genome sequence analysis revealed a number of non-synonymous mutations between copies of the kinetochore genes from Festuca and Lolium genomes. Furthermore, we found that outer kinetochore proteins NDC80 and NNF1 were exclusively expressed from the Lolium allele. We hypothesize that silencing of Festuca alleles results in improper attachment of Festuca chromosomes to karyokinetic spindle and subsequently their gradual elimination.ISSN:0028-646XISSN:1469-813

Cytonuclear interplay in auto- and allopolyploids: a multifaceted perspective from the Festuca-Lolium complex

Restoring cytonuclear stoichiometry is necessary after whole-genome duplication (WGD) and interspecific/intergeneric hybridization in plants. We investigated this phenomenon in auto- and allopolyploids of the Festuca-Lolium complex providing insights into the mechanisms governing cytonuclear interactions in early polyploid and hybrid generations. Our study examined the main processes potentially involved in restoring the cytonuclear balance after WGD comparing diploids and new and well-established autopolyploids. We uncovered that both the number of chloroplasts and the number of chloroplast genome copies were significantly higher in the newly established autopolyploids and grew further in more established autopolyploids. The increase in the copy number of the chloroplast genome exceeded the rise in the number of chloroplasts and fully compensated for the doubling of the nuclear genome. In addition, changes in nuclear and organelle gene expression were insignificant. Allopolyploid Festuca × Lolium hybrids displayed potential structural conflicts in parental protein variants within the cytonuclear complexes. While biased maternal allele expression has been observed in numerous hybrids, our results suggest that its role in cytonuclear stabilization in the Festuca × Lolium hybrids is limited. This study provides insights into the restoration of the cytonuclear stoichiometry, yet it emphasizes the need for future research to explore post-transcriptional regulation and its impact on cytonuclear gene expression stoichiometry. Our findings may enhance the understanding of polyploid plant evolution, with broader implications for the study of cytonuclear interactions in diverse biological contexts.This article is published as Shahbazi, Mehrdad, Joanna Majka, Denisa Kubíková, Zbigniew Zwierzykowski, Marek Glombik, Jonathan F. Wendel, Joel Sharbrough et al. "Cytonuclear interplay in auto‐and allopolyploids: a multifaceted perspective from the Festuca‐Lolium complex." The Plant Journal (2024). doi:10.1111/tpj.16659. © 2024 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made