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Nitrogen (N) is a critical element for plant growth and development. Hence, improving nitrogen use efficiency (NUE) is vital for reducing costs and the environmental impact of agricultural practices. Understanding the genetic control of N metabolism is crucial to improve NUE, especially in agronomically important plants, such as barley (Hordeum vulgare). Using bioinformatics and functional genomics tools, we identified and characterized sixteen barley nitrogen metabolism-related gene families (HvNMGs) on a genome-wide scale, analysing gene features and evolution. These genes, located on six of seven barley chromosomes, are highly conserved in plants (including barley, rice, and Arabidopsis), as shown by phylogenetic analysis. We further explored the evolutionary relationships of NMGs through a genome-to-genome synteny analysis, which indicated higher conservation of NMGs between barley and other monocots, suggesting that these orthologous pairs predate species divergence. Protein-protein interaction analyses revealed that all of the HvNMGs show interactions, mainly with each other. The H. vulgare miRNAs target sites (hvu-miR) prediction identified six hvu-miR in 4 HvNMGs (HvGABA-T2, HvALDH10-1, HvALDH10-2 and HvARGAH), indicating their potential involvement in stress responses. The expression patterns analysis of publicly available RNA-seq data revealed that HvNMGs are expressed in all developmental stages of barley, and they respond to different stress conditions, indicating their essential role in plant growth, development and stress response. The organ-specific expression analysis, conducted using qPCR, of HvNMGs revealed higher expression of HvNiR and HvNRs in the leaf and significantly higher expression of HvARGAH and HvALDH10 in the spike than in other tissues, showing that some of the genes may be particularly important in some tissues than others. This data provides a foundation for understanding HvNMG function and could be used to improve barley yield by enhancing NUE - an important goal for both crop productivity and environmental sustainability., Competing Interests: Declaration of competing interest None Declared., (Copyright © 2024. Published by Elsevier Ltd.)

Genetic improvements of solanaceous crops for quality and stress responsive traits are needed because of the central role vegetables and fruits have in providing nutrients to human diets. Copper amine oxidase (CuAO) encoding genes involved in metabolism of primary/di-amine nitrogenous compounds, play a role in balancing internal nitrogen (N) pools especially when external N supply fluctuates during growth, development and environmental stresses. In the present study, we investigated the occurrence, molecular evolution and possible role(s) of these unknown genes in tomato crops. Multiple genome-wide bioinformatics approaches led to the identification of eight bona fide CuAO genes (SlCuAO1-SlCuAO8) in the tomato genome with gene numbers like those in Arabidopsis and rice indicating their conserved functional relevance with a tandemly duplicated SlCuAO6-SlCuAO7 pair at chr.9. A conserved intron-exon size and phase distribution for SlCuAO2, 3, 4 pairs are similar to a recently identified single duckweed SpCuAO1 orthologue gene indicating its evolutionary conservation. Synteny analysis showed their closest association to Arabidopsis and but not with rice. Transcriptome data indicated that gene expression for about six genes (SlCuAO1, 2, 3, 4, 6, 7) is root specific, fruit specific for SlCuAO5 and flower specific for SlCuAO8 thus indicating amine oxidation is variable across tissues with a prominance in the root tissue. The majority of CuAO genes are negatively regulated by methyl jasmonate. Positive regulation, however, involves CuAO3/8. Transcript analysis of the ethylene-deficient transgenic lines indicated that ethylene is required for activation of SlCuAO4. CuAO4 and CuAO5 exhibited most significant tissues-independent gene expression responses across various nitrogen regimes. Drought, heat and N stress identified CuAO5 as an overlapping highly expressed gene that corroborates with putrescine accumulation for free and conjugated forms with an opposite abundance of bound forms. Taken together our study highlights new insights into the roles of copper amine oxidation genes and identifies CuAO5 as a multiple stress induced gene that can be used in genetic improvement programs for combining heat, drought and nitrogen use efficiency related traits., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier GmbH.)

Considering that maize ( Zea mays L.) is a staple food for a large segment of the population worldwide, many attempts have been made to improve the nutritional value of its grain and at the same time to achieve sustainable cropping systems. The present study aimed to characterize the composition and nutritional value of maize grain as influenced by cropping system, genetic background (variety), and growing year using untargeted NMR metabolomics. The composition of both water- (sugars and polyols, organic acids, and amino acids) and liposoluble metabolites (free and esterified fatty acids, sterols, and lipids) extracted from the maize grain was determined. Multivariate statistical analyses (PCA and ANOVA) pointed to the growing year and the variety as the most important random and fixed factors, respectively, influencing the metabolite profile. The samples were separated along PC1 and PC3 according to the growing year and the variety, respectively. A higher content of citric acid and diunsaturated fatty acids and a lower content of tyrosine, trigonelline, and monounsaturated fatty acids was observed in the organic with respect to the conventional variety. The effect of the cropping system was overwhelmed by the random effect of the growing year. The results provide novel knowledge on the influence of agronomic practices on maize micronutrient contents.

Main Conclusion: Molecular mechanisms of biological rhythms provide opportunities to harness functional allelic diversity in core (and trait- or stress-responsive) oscillator networks to develop more climate-resilient and productive germplasm. The circadian clock senses light and temperature in day-night cycles to drive biological rhythms. The clock integrates endogenous signals and exogenous stimuli to coordinate diverse physiological processes. Advances in high-throughput non-invasive assays, use of forward- and inverse-genetic approaches, and powerful algorithms are allowing quantitation of variation and detection of genes associated with circadian dynamics. Circadian rhythms and phytohormone pathways in response to endogenous and exogenous cues have been well documented the model plant Arabidopsis. Novel allelic variation associated with circadian rhythms facilitates adaptation and range expansion, and may provide additional opportunity to tailor climate-resilient crops. The circadian phase and period can determine adaptation to environments, while the robustness in the circadian amplitude can enhance resilience to environmental changes. Circadian rhythms in plants are tightly controlled by multiple and interlocked transcriptional-translational feedback loops involving morning (CCA1, LHY), mid-day (PRR9, PRR7, PRR5), and evening (TOC1, ELF3, ELF4, LUX) genes that maintain the plant circadian clock ticking. Significant progress has been made to unravel the functions of circadian rhythms and clock genes that regulate traits, via interaction with phytohormones and trait-responsive genes, in diverse crops. Altered circadian rhythms and clock genes may contribute to hybrid vigor as shown in Arabidopsis, maize, and rice. Modifying circadian rhythms via transgenesis or genome-editing may provide additional opportunities to develop crops with better buffering capacity to environmental stresses. Models that involve clock gene‒phytohormone‒trait interactions can provide novel insights to orchestrate circadian rhythms and modulate clock genes to facilitate breeding of all season crops., (© 2024. The Author(s).)

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N 6 -(Δ 2 -isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N 6 -(Δ 2 -isopentenyl)adenosine-5'-monophosphate (iPRMP) → N 6 -(Δ 2 -isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Masson SAS.)

L-Ascorbic acid (AsA), a strong antioxidant, serves as an enzyme cofactor and redox status marker, modulating a plethora of biological processes. As tomato commercial varieties and hybrids possess relatively low amounts of AsA, the improvement of fruit AsA represents a strategic goal for enhanced human health. Previously, we have suggested that GDP-L-Galactose phosphorylase (GGP) and L-galactose-1-phosphate phosphatase (GPP) can serve as possible targets for AsA manipulation in tomato (Solanum lycopersicon L.) fruit. To this end, we produced and evaluated T3 transgenic tomato plants carrying these two genes under the control of CaMV-35S and two fruit specific promoters, PPC2 and PG-GGPI. The transgenic lines had elevated levels of AsA, with the PG-GGP1 line containing 3-fold more AsA than WT, without affecting fruit characteristics. Following RNA-Seq analysis, 164 and 13 DEGs were up- or down-regulated, respectively, between PG-GGP1 and WT pink fruits. PG-GGP1 fruit had a distinct number of up-regulated transcripts associated with cell wall modification, ethylene biosynthesis and signaling, pollen fertility and carotenoid metabolism. The elevated AsA accumulation resulted in the up regulation of AsA associated transcripts and alternative biosynthetic pathways suggesting that the entire metabolic pathway was influenced, probably via master regulation. We show here that AsA-fortification of tomato ripe fruit via GGP1 overexpression under the action of a fruit specific promoter PG affects fruit development and ripening, reduces ethylene production, and increased the levels of sugars, and carotenoids, supporting a robust database to further explore the role of AsA induced genes for agronomically important traits, breeding programs and precision gene editing approaches., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

We explored the polyamine (PA) metabolic pathway genes in barley (Hv) to understand plant development and stress adaptation in Gramineae crops with emphasis on leaf senescence. Bioinformatics and functional genomics tools were utilized for genome-wide identification, comprehensive gene features, evolution, development and stress effects on the expression of the polyamine metabolic pathway gene families (PMGs). Three S-adenosylmethionine decarboxylases (HvSAMDCs), two ornithine decarboxylases (HvODCs), one arginine decarboxylase (HvADC), one spermidine synthase (HvSPDS), two spermine synthases (HvSPMSs), five copper amine oxidases (HvCuAOs) and seven polyamine oxidases (HvPAOs) members of PMGs were identified and characterized in barley. All the HvPMG genes were found to be distributed on all chromosomes of barley. The phylogenetic and comparative assessment revealed that PA metabolic pathway is highly conserved in plants and the prediction of nine H. vulgare miRNAs (hvu-miR) target sites, 18 protein-protein interactions and 961 putative CREs in the promoter region were discerned. Gene expression of HvSAMDC3, HvCuAO7, HvPAO4 and HvSPMS1 was apparent at every developmental stage. SPDS/SPMS gene family was found to be the most responsive to induced leaf senescence. This study provides a reference for the functional investigation of the molecular mechanism(s) that regulate polyamine metabolism in plants as a tool for future breeding decision management systems., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The photosystem II (PSII) reaction centre is the critical supramolecular pigment-protein complex in the chloroplast which catalyses the light-induced transfer of electrons from water to plastoquinone. Structural studies have demonstrated the existence of an oligomeric PSII. We carried out radiation inactivation target analysis (RTA), together with sucrose gradient ultracentrifugation (SGU) of PSII, to study the functional size of PSII in diverse plant species under physiological and stress conditions. Two PSII populations, made of dimeric and monomeric core particles, were revealed in Pisum sativum , Spinacea oleracea , Phaseulus vulgaris , Medicago sativa , Zea mais and Triticum durum . However, this core pattern was not ubiquitous in the higher plants since we found one monomeric core population in Vicia faba and a dimeric core in the Triticum durum yellow-green strain, respectively. The PSII functional sizes measured in the plant seedlings in vivo, as a decay of the maximum quantum yield of PSII for primary photochemistry, were in the range of 75-101 ± 18 kDa, 2 to 3 times lower than those determined in vitro. Two abiotic stresses, heat and drought, imposed individually on Pisum sativum , increased the content of the dimeric core in SGU and the minimum functional size determined by RTA in vivo. These data suggest that PSII can also function as a monomer in vivo, while under heat and drought stress conditions, the dimeric PSII structure is predominant.

Malnutrition, unhealthy diets, and lifestyle changes have become major risk factors for non-communicable diseases while adversely impacting economic growth and sustainable development. Anthocyanins, a group of flavonoids that are rich in fruits and vegetables, contribute positively to human health. This review focuses on genetic variation harnessed through crossbreeding and biotechnology-led approaches for developing anthocyanins-rich fruit and vegetable crops. Significant progress has been made in identifying genes involved in anthocyanin biosynthesis in various crops. Thus, the use of genetics has led to the development and release of anthocyanin-rich potato and sweet potato cultivars in Europe and the USA. The purple potato 'Kufri Neelkanth' has been released for cultivation in northern India. In Europe, the anthocyanin-rich tomato cultivar 'Sun Black' developed via the introgression of Aft and atv genes has been released. The development of anthocyanin-rich food crops without any significant yield penalty has been due to the use of genetic engineering involving specific transcription factors or gene editing. Anthocyanin-rich food ingredients have the potential of being more nutritious than those devoid of anthocyanins. The inclusion of anthocyanins as a target characteristic in breeding programs can ensure the development of cultivars to meet the nutritional needs for human consumption in the developing world.

Main Conclusion: Identification of the polyamine biosynthetic pathway genes in duckweed S. polyrhiza reveals presence of prokaryotic as well as land plant-type ADC pathway but absence of ODC encoding genes. Their differential gene expression and transcript abundance is shown modulated by exogenous methyl jasmonate, salinity, and acidic pH. Genetic components encoding for polyamine (PA) biosynthetic pathway are known in several land plant species; however, little is known about them in aquatic plants. We utilized recently sequenced three duckweed (Spirodela polyrhiza) genome assemblies to map PA biosynthetic pathway genes in S. polyrhiza. PA biosynthesis in most higher plants except for Arabidopsis involves two pathways, via arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). ADC-mediated PA biosynthetic pathway genes, namely, one arginase (SpARG1), two arginine decarboxylases (SpADC1, SpADC2), one agmatine iminohydrolase/deiminase (SpAIH), one N-carbamoyl putrescine amidase (SpCPA), three S-adenosylmethionine decarboxylases (SpSAMDc1, 2, 3), one spermidine synthase (SpSPDS1) and one spermine synthase (SpSPMS1) in S. polyrhiza genome were identified here. However, no locus was found for ODC pathway genes in this duckweed. Hidden Markov Model protein domain analysis established that SpADC1 is a prokaryotic/biodegradative type ADC and its molecular phylogenic classification fell in a separate prokaryotic origin ADC clade with SpADC2 as a biosynthetic type of arginine decarboxylase. However, thermospermine synthase (t-SPMS)/Aculis5 genes were not found present. Instead, one of the annotated SPDS may also function as SPMS, since it was found associated with the SPMS phylogenetic clade along with known SPMS genes. Moreover, we demonstrate that S. polyrhiza PA biosynthetic gene transcripts are differentially expressed in response to unfavorable conditions, such as exogenously added salt, methyl jasmonate, or acidic pH environment as well as in extreme temperature regimes. Thus, S. polyrhiza genome encodes for complete polyamine biosynthesis pathway and the genes are transcriptionally active in response to changing environmental conditions suggesting an important role of polyamines in this aquatic plant., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Polyamines have been implicated in ameliorating the detrimental effects of drought and saline conditions on plant growth and development. The independent impact of these two abiotic stresses on polyamine (PA) biosynthesis, catabolism, and homeostasis, as well as on their transcript abundance in tomato leaves, is presented here. We show that the total levels of putrescine (PUT), spermidine (SPD), and spermine (SPM) increase up to 72 h during drought and up to 48 h during salinity stress before their precipitable drop thereafter. Thus, tomato plants maintain survivability to drought as well as salinity stress for up to 3 and 2 days, respectively. Independent multivariant analyses of drought and salinity stress kinetic data separately showed a closer association with levels of free, conjugated, and bound forms of SPD and SPM, but not with free or bound PUT. However, combined multivariant analyses showed a closer association of free SPD, conjugated SPD, and bound SPD with both stresses; SPD-bound and SPM conjugated with drought; and free SPM and conjugated PUT with salinity stress, respectively. PA biosynthesis genes, ARG1 , SPDS1 , and SAMDc3 , segregated with drought and SPDS2 with salinity stress. PA catabolic genes CuAO4-like and PAO4 were associated with drought and salinity stresses, respectively, suggesting differential involvement of PA biosynthesis and catabolic genes in drought and salinity stresses. Pearson correlation indicated mostly positive correlations between the levels of free, conjugated, and bound forms of PUT, SPD, and SPM under drought and salinity stress. However, negative correlations were mostly seen between the levels of various forms of the PAs and their biosynthesis/catabolic genes. Levels of different PA forms had a twofold higher negative correlation during drought as compared to salinity stress (66 vs. 32) and with transcript levels of PA biosynthesis and catabolic genes. Transcripts of light-harvesting chlorophyll a/b-binding genes were generally positively associated with different forms of PAs but negatively to carbon flow genes. Most of the PA biosynthesis genes were coordinately regulated under both stresses. Collectively, these results indicate that PAs are distinctly regulated under drought and salinity stress with different but specific homologs of PA biosynthesis and catabolic genes contributing to the accumulation of free, conjugated, and bound forms of PAs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Upadhyay, Fatima, Handa and Mattoo.)

Symbiotic nitrogen fixation (SNF) process makes legume crops self-sufficient in nitrogen (N) in sharp contrast to cereal crops that require an external input by N-fertilizers. Since the latter process in cereal crops results in a huge quantity of greenhouse gas emission, the legume production systems are considered efficient and important for sustainable agriculture and climate preservation. Despite benefits of SNF, and the fact that chemical N-fertilizers cause N-pollution of the ecosystems, the focus on improving SNF efficiency in legumes did not become a breeder's priority. The size and stability of heritable effects under different environment conditions weigh significantly on any trait useful in breeding strategies. Here we review the challenges and progress made toward decoding the heritable components of SNF, which is considerably more complex than other crop allelic traits since the process involves genetic elements of both the host and the symbiotic rhizobial species. SNF-efficient rhizobial species designed based on the genetics of the host and its symbiotic partner face the test of a unique microbiome for its success and productivity. The progress made thus far in commercial legume crops with relevance to the dynamics of host-rhizobia interaction, environmental impact on rhizobial performance challenges, and what collectively determines the SNF efficiency under field conditions are also reviewed here., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Goyal, Mattoo and Schmidt.)

Lipoxygenases (LOXs) (EC 1.13.11.12) catalyze the oxygenation of fatty acids and produce oxylipins, including the plant hormone jasmonic acid (JA) and its methyl ester, methyl jasmonate (MeJA). Little information is available about the LOX gene family in aquatic plants. We identified a novel LOX gene family comprising nine LOX genes in the aquatic plant Spirodela polyrhiza (greater duckweed). The reduced anatomy of S. polyrhiza did not lead to a reduction in LOX family genes. The 13-LOX subfamily, with seven genes, predominates, while the 9-LOX subfamily is reduced to two genes, an opposite trend from known LOX families of other plant species. As the 13-LOX subfamily is associated with the synthesis of JA/MeJA, its predominance in the Spirodela genome raises the possibility of a higher requirement for the hormone in the aquatic plant. JA-/MeJA-based feedback regulation during culture aging as well as the induction of LOX gene family members within 6 h of salt exposure are demonstrated.

Ripening of tomato fruit leads, in general, to a sequential decrease in the endogenous levels of polyamines spermidine (SPD) and spermine (SPM), while the trend for the diamine putrescine (PUT) levels is generally an initial decrease, followed by a substantial increase, and thereafter reaching high levels at the red ripe fruit stage. However, genetic engineering fruit-specific expression of heterologous yeast S-adenosylmethionine (SAM) decarboxylase in tomato has been found to result in a high accumulation of SPD and SPM at the cost of PUT. This system enabled a genetic approach to determine the impact of increased endogenous levels of biogenic amines SPD and SPM in tomato (579HO transgenic line) and on the biogenesis, transcription, processing, and stability of ribosomal RNA (rRNA) genes in tomato fruit as compared with the non-transgenic 556AZ line. One major biogenetic process regulating transcription and processing of pre-mRNA complexes in the nucleus involves small nucleolar RNAs (snoRNAs). To determine the effect of high levels of SPD and SPM on these latter processes, we cloned, sequenced, and identified a box C/D snoRNA cluster in tomato, namely, SlSnoR12 , SlU24a , Slz44a , and Slz132b . Similar to this snoRNA cluster housed on chromosome (Chr.) 6, two other noncoding C/D box genes, SlsnoR12.2 and SlU24b, with a 94% identity to those on Chr. 6 were found located on Chr. 3. We also found that other snoRNAs divisible into snoRNA subclusters A and B, separated by a uridine rich spacer, were decorated with other C/D box snoRNAs, namely, J10.3, Z131a/b, J10.1, and Z44a, followed by z132a, J11.3, z132b, U24, Z20, U24a, and J11. Several of these, for example, SlZ44a , Slz132b, and SlU24a share conserved sequences similar to those in Arabidopsis and rice. RNAseq analysis of high SPD/SPM transgenic tomatoes (579HO line) showed significant enrichment of RNA polymerases, ribosomal, and translational protein genes at the breaker+8 ripening stage as compared with the 556AZ control. Thus, these results indicate that SPD/SPM regulates snoRNA and rRNA expression directly or indirectly, in turn, affecting protein synthesis, metabolism, and other cellular activities in a positive manner.

Polyamines (PAs) regulate growth in plants and modulate the whole plant life cycle. They have been associated with different abiotic and biotic stresses, but little is known about the molecular regulation involved. We quantified gene expression of PA anabolic and catabolic pathway enzymes in tomato ( Solanum lycopersicum cv. Ailsa Craig) leaves under heat versus cold stress. These include arginase 1 and 2 , arginine decarboxylase 1 and 2 , agmatine iminohydrolase / deiminase 1 , N-carbamoyl putrescine amidase , two ornithine decarboxylases , three S-adenosylmethionine decarboxylases , two spermidine synthases ; spermine synthase ; flavin-dependent polyamine oxidases ( SlPAO4-like and SlPAO2 ) and copper dependent amine oxidases ( SlCuAO and SlCuAO-like ). The spatiotemporal transcript abundances using qRT-PCR revealed presence of their transcripts in all tissues examined, with higher transcript levels observed for SAMDC1 , SAMDC2 and ADC2 in most tissues. Cellular levels of free and conjugated forms of putrescine and spermidine were found to decline during heat stress while they increased in response to cold stress, revealing their differential responses. Transcript levels of ARG2 , SPDS2 , and PAO4-like increased in response to both heat and cold stresses. However, transcript levels of ARG1/2 , AIH1 , CPA , SPDS1 and CuAO4 increased in response to heat while those of ARG2, ADC1,2, ODC1, SAMDC1,2,3, PAO2 and CuPAO4-like increased in response to cold stress, respectively. Transcripts of ADC1,2 , ODC1,2 , and SPMS declined in response to heat stress while ODC2 transcripts declined under cold stress. These results show differential expression of PA metabolism genes under heat and cold stresses with more impairment clearly seen under heat stress. We interpret these results to indicate a more pronounced role of PAs in cold stress acclimation compared to that under heat stress in tomato leaves.

Heat shock proteins (HSPs) are ubiquitous and highly conserved in nature. Heat stress upregulates their gene expression and now it is known that they are also developmentally regulated. We have studied regulation of small HSP genes during ripening of tomato fruit. In this study, we identify two small HSP genes, SlHSP17.7A and SlHSP17.7B , localized on tomato Chr.6 and Chr.9, respectively. Each gene encodes proteins constituting 154 amino acids and has characteristic domains as in other sHSP genes. We found that SlHSP17.7A and SlHSP17.7B gene expression is low in the vegetative tissues as compared to that in the fruit. These sHSP genes are characteristically expressed in a fruit-ripening fashion, being upregulated during the ripening transition of mature green to breaker stage. Their expression patterns mirror that of the rate-limiting ethylene biosynthesis gene ACC (1-aminocyclopropane-1-carboxylic acid) synthase, SlACS2, and its regulator SlMADS-RIN . Exogenous application of ethylene to either mature green tomato fruit or tomato leaves suppressed the expression of both the SlHSP17.7A, B genes. Notably and characteristically, a transgenic tomato line silenced for SlACS2 gene and whose fruits produce ~50% less ethylene in vivo , had higher expression of both the sHSP genes at the fruit ripening transition stages [breaker (BR) and BR+3] than the control fruit. Moreover, differential gene expression of SlHSP17.7A versus SlHSP17.7B gene was apparent in the tomato ripening mutants- rin/rin , nor/nor , and Nr/Nr , with the expression of SlHSP17.7A being significantly reduced but that of SlHSP17.7B significantly upregulated as compared to the wild type (WT). These data indicate that ethylene negatively regulates transcriptional abundance of both these sHSPs. Transient overexpression of the ripening regulator SlMADS-RIN in WT and ACS2-AS mature green tomato fruits suppressed the expression of SlHSP17.7A but not that of SlHSP17.7B . Thus, ethylene directly or in tune with SlMADS-RIN regulates the transcript abundance of both these sHSP genes., (Copyright © 2020 Upadhyay, Tucker and Mattoo.)

Shape and size are important features of fruits. Studies using tomatoes expressing yeast Spermidine Synthase under either a constitutive or a fruit-ripening promoter showed obovoid fruit phenotype compared to spherical fruit in controls, suggesting that polyamines (PAs) have a role in fruit shape. The obovoid fruit pericarp exhibited decreased cell layers and pericarp thickness compared to wild-type fruit. Transgenic floral buds and ovaries accumulated higher levels of free PAs, with the bound form of PAs being predominant. Transcripts of the fruit shape genes, SUN1 and OVATE , and those of CDKB2 , CYCB2 , KRP1 and WEE1 genes increased significantly in the transgenic ovaries 2 and 5 days after pollination (DAP). The levels of cell expansion genes CCS52A / B increased at 10 and 20 DAP in the transgenic fruits and exhibited negative correlation with free or bound forms of PAs. In addition, the cell layers and pericarp thickness of the transgenic fruits were inversely associated with free or bound PAs in 10 and 20 DAP transgenic ovaries. Collectively, these results provide evidence for a linkage between PA homeostasis and expression patterns of fruit shape, cell division, and cell expansion genes during early fruit development, and suggest role(s) of PAs in tomato fruit architecture.

Polyamines (PAs) constituting putrescine (Put), spermidine (Spd), and spermine (Spm) are ubiquitous in all organisms and play essential roles in the growth and developmental processes in living organisms, including plants. Evidences obtained through genetic, biochemical, and transgenic approaches suggest a tight homeostasis for cellular PA levels. Altered cellular PA homeostasis is associated with abnormal phenotypes. However, the mechanisms involved for these abnormalities are not yet fully understood, nor is it known whether cellular ratios of different polyamines play any role(s) in specific plant processes. We expressed a yeast spermidine synthase gene ( ySpdSyn ) under a constitutive promoter CaMV35S in tomato and studied the different phenotypes that developed. The constitutive expression of ySpdSyn resulted in variable flower phenotypes in independent transgenic lines, some of which lacked fruit and seed set. Quantification of PA levels in the developing flowers showed that the transgenic plants without fruit and seed set had significantly reduced Spd levels as well as low Spd/Put ratio compared to the transgenic lines with normal fruit and seed set. Transcript levels of SlDELLA , GA-20oxidase-1 , and GA-3oxidase-2 , which impact gibberellin (GA) metabolism and signaling, were significantly reduced in bud tissue of transgenic lines that lacked fruit and seed set. These findings indicate that PAs, particularly Spd, impact floral organ identity and fruit set in tomato involving GA metabolism and signaling. Furthermore, we suggest that a nexus exists between PA ratios and developmental programs in plants., (Copyright © 2019 Nambeesan, Mattoo and Handa.)

Lipoxygenases (LOXs; EC 1.13.11.12) catalyze the oxygenation of fatty acids to produce oxylipins including the jasmonate family of plant hormones. The involvement of jasmonates in plant growth and development and during abiotic stress has been documented, however, the response and regulation of each member of the LOX gene family under various abiotic stresses is yet to be fully deciphered. Previously, we identified fourteen members of the tomato LOX gene family, which were divisible into nine genes representing the 9-LOX family members and five others representing the 13-LOX family members based on the carbon oxidation position specificity of polyunsaturated fatty acids. Here, we have determined the transcript abundance patterns of all the 14 LOX genes in response to four independent abiotic stresses, namely, heat, cold, drought and salt. Our results show that each of these stresses leads to a time-dependent, variable or indifferent response of specific and different set(s) of LOX gene members of both subfamilies, differentiating functional relevance of the 14 LOX genes analyzed. Out of the 14 gene members, three LOX genes were expressed constitutively or were non-responsive to either heat (SlLOX9), cold (SlLOX9) or salt (SlLOX4) stress. An in-silico LOX gene promoter search for stress-responsive elements revealed that only some but not all of the LOX genes indeed are decorated with specific and known stress responsive cis-acting elements. Thus, these data implicate some other, yet to be discovered, cis-acting elements present in the LOX gene family members, which seemingly regulate tomato responses to defined abiotic stresses presented here., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Polyamines (PAs) are low molecular weight organic cations comprising biogenic amines that play multiple roles in plant growth and senescence. PA metabolism was found to play a central role in metabolic and genetic reprogramming during dark-induced barley leaf senescence (DILS). Robust PA catabolism can impact the rate of senescence progression in plants. We opine that deciphering senescence-dependent polyamine-mediated multidirectional metabolic crosstalks is important to understand regulation and involvement of PAs in plant death and re-mobilization of nutrients during senescence. This will involve optimizing the use of PA biosynthesis inhibitors, robust transgenic approaches to modulate PA biosynthetic and catabolic genes, and developing novel germplasm enriched in pro- and anti-senescence traits to ensure sustained crop productivity. PA-mediated delay of senescence can extend the photosynthesis capacity, thereby increasing grain starch content in malting grains such as barley. On the other hand, accelerating the onset of senescence can lead to increases in mineral and nitrogen content in grains for animal feed. Unraveling the "polyamine metabolic switch" and delineating the roles of PAs in senescence should further our knowledge about autophagy mechanisms involved in plant senescence as well as mammalian systems. It is noteworthy that inhibitors of PA biosynthesis block cell viability in animal model systems (cell tumor lines) to control some cancers, in this instance, proliferative cancer cells were led toward cell death. Likewise, PA conjugates work as signal carriers for slow release of regulatory molecule nitric oxide in the targeted cells. Taken together, these and other outcomes provide examples for developing novel therapeutics for human health wellness as well as developing plant resistance/tolerance to stress stimuli.

Lipoxygenases (LOXs) (EC 1.13.11.12) catalyze the oxygenation of fatty acids and produce oxylipins including the plant hormone jasmonate (jasmonic acid/methyl jasmonate; MeJA). Little is known about the tomato LOX gene family members that impact tomato growth and development, and less so about their feed-back regulation in response to MeJA. We present genome wide identification of 14 LOX gene family members in tomato which map unevenly on 12 chromosomes. The characteristic structural features of 9-LOX and 13-LOX tomato gene family, their protein domains/features, and divergence are presented. Quantification of the expression patterns of all the 14 SlLOX gene members segregated the members based on differential association with growth, development, or fruit ripening. We also identified those SlLOX genes whose transcription responds to exogenous MeJA and/or wounding stress. MeJA-based feedback regulation that involves activation of specific members of LOX genes is defined. Specific nature of SlLOX gene regulation in tomato is defined. The novel data on dynamics of SlLOX gene expression should help catalyze future strategies to elucidate role(s) of each gene member in planta and for crop biotechnological intervention., (Published by Elsevier GmbH.)

Photosystem II (PSII) reaction centre D1 protein of oxygenic phototrophs is pivotal for sustaining photosynthesis. Also, it is targeted by herbicides and herbicide-resistant weeds harbour single amino acid substitutions in D1. Conservation of D1 primary structure is seminal in the photosynthetic performance in many diverse species. In this study, we analysed built-in and environmentally-induced (high temperature and high photon fluency - HT/HL) phenotypes of two D1 mutants of Chlamydomonas reinhardtii with Ala250Arg (A250R) and Ser264Lys (S264K) substitutions. Both mutations differentially affected efficiency of electron transport and oxygen production. In addition, targeted metabolomics revealed that the mutants undergo specific differences in primary and secondary metabolism, namely, amino acids, organic acids, pigments, NAD, xanthophylls and carotenes. Levels of lutein, β-carotene and zeaxanthin were in sync with their corresponding gene transcripts in response to HT/HL stress treatment in the parental (IL) and A250R strains. D1 structure analysis indicated that, among other effects, remodelling of H-bond network at the Q B site might underpin the observed phenotypes. Thus, the D1 protein, in addition to being pivotal for efficient photosynthesis, may have a moonlighting role in rewiring of specific metabolic pathways, possibly involving retrograde signalling.

Barley crop model was analyzed for early and late events during the dark-induced leaf senescence (DILS) as well as for deciphering critical time limit for reversal of the senescence process. Chlorophyll fluorescence vitality index Rfd was determined as the earliest parameter that correlated well with the cessation of photosynthesis prior to microautophagy symptoms, initiation of DNA degradation, and severalfold increase in the endonuclease BNUC1. DILS was found characterized by up-regulation of processes that enable recycling of degraded macromolecules and metabolites, including increased NH 4 + remobilization, gluconeogenesis, glycolysis, and partial up-regulation of glyoxylate and tricarboxylate acid cycles. The most evident differences in gene medleys between DILS and developmental senescence included hormone-activated signaling pathways, lipid catabolic processes, carbohydrate metabolic processes, low-affinity ammonia remobilization, and RNA methylation. The mega-autophagy symptoms were apparent much later, specifically on day 10 of DILS, when disruption of organelles-nucleus and mitochondria -became evident. Also, during this latter-stage programmed cell death processes, namely, shrinking of the protoplast, tonoplast interruption, and vacuole breakdown, chromatin condensation, more DNA fragmentation, and disintegration of the cell membrane were prominent. Reversal of DILS by re-exposure of the plants from dark to light was possible until but not later than day 7 of dark exposure and was accompanied by regained photosynthesis, increase in chlorophyll, and reversal of Rfd, despite activation of macro-autophagy-related genes., (© 2018 American Society of Plant Biologists. All rights reserved.)

Retrograde signaling is an intracellular communication process defined by cues generated in chloroplast and mitochondria which traverse membranes to their destination in the nucleus in order to regulate nuclear gene expression and protein synthesis. The coding and decoding of such organellar message(s) involve gene medleys and metabolic components about which more is known in higher plants than the unicellular organisms such as algae. Chlamydomonas reinhardtii is an oxygenic microalgal model for genetic and physiological studies. It harbors a single chloroplast and is amenable for generating mutants. The focus of this review is on studies that delineate retrograde signaling in Chlamydomonas vis a vis higher plants. Thus, communication networks between chloroplast and nucleus involving photosynthesis- and ROS-generated signals, functional tetrapyrrole biosynthesis intermediates, and Ca 2+ -signaling that modulate nuclear gene expression in this alga are discussed. Conceptually, different signaling components converge to regulate either the same or functionally-overlapping gene products., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Biogenic amines-polyamines (PAs), particularly putrescine, spermidine and spermine are ubiquitous in all living cells. Their indispensable roles in many biochemical and physiological processes are becoming commonly known, including promoters of plant life and differential roles in human health and disease. PAs positively impact cellular functions in plants-exemplified by increasing longevity, reviving physiological memory, enhancing carbon and nitrogen resource allocation/signaling, as well as in plant development and responses to extreme environments. Thus, one or more PAs are commonly found in genomic and metabolomics studies using plants, particulary during different abiotic stresses. In humans, a general decline in PA levels with aging occurs parallel with some human health disorders. Also, high PA dose is detrimental to patients suffering from cancer, aging, innate immunity and cognitive impairment during Alzheimer and Parkinson diseases. A dichotomy exists in that while PAs may increase longevity and reduce some age-associated cardiovascular diseases, in disease conditions involving higher cellular proliferation, their intake has negative consequences. Thus, it is essential that PA levels be rigorously quantified in edible plant sources as well as in dietary meats. Such a database can be a guide for medical experts in order to recommend which foods/meats a patient may consume and which ones to avoid. Accordingly, designing both high and low polyamine diets for human consumption are in vogue, particularly in medical conditions where PA intake may be detrimental, for instance, cancer patients. In this review, literature data has been collated for the levels of the three main PAs, putrescine, spermidine and spermine, in different edible sources-vegetables, fruits, cereals, nuts, meat, sea food, cheese, milk, and eggs. Based on our analysis of vast literature, the effects of PAs in human/animal health fall into two broad, Yang and Yin, categories: beneficial for the physiological processes in healthy cells and detrimental under pathological conditions.

Clustered class-I small heat-shock protein (sHSP) chaperone genes, SlHSP17.6, SlHSP20.0 and SlHSP20.1, in tomato are demonstrated to be transcriptionally regulated by ethylene during mature green (MG) fruit transition into ripening. These genes are constitutively expressed at MG fruit stage in two different tomato genotypes as well as in their ripening mutants, including rin, nor and Nr, and an ethylene-deficient transgenic line, ACS2-antisense. Notably, ethylene treatment of the MG fruit led to significant sHSP gene suppression in both wild-types, ACS2-antisense, nor/nor and Nr/Nr, but not the rin/rin mutant. Inability of ethylene to suppress sHSP genes in rin/rin mutant, which harbors MADS-RIN gene mutation, suggests that MADS-RIN transcription factor regulates the expression of these genes. Treatment of the wild type and ACS2-antisense fruit with the ethylene-signaling inhibitor, 1-methylcyclopropane (1-MCP), reversed the sHSP gene suppression. Transcripts of representative ethylene-responsive and ripening-modulated genes confirmed and validated sHSP transcript profile patterns. In silico analysis in conjunction with chromatin immunoprecipitation demonstrated MADS-RIN protein binding to specific CArG motifs present in the promoters of these chaperone genes. The results establish MADS-RIN protein as a transcriptional regulator of these chaperone genes in an ethylene-dependent manner, and that MADS-RIN protein-regulation of sHSPs is integral to tomato fruit ripening.

Plants execute an array of mechanisms in response to stress which include upregulation of defense-related proteins and changes in specific metabolites. Polyamines - putrescine (Put), spermidine (Spd), and spermine (Spm) - are metabolites commonly found associated with abiotic stresses such as chilling stress. We have generated two transgenic tomato lines (556HO and 579HO) that express yeast S-adenosylmethionine decarboxylase and specifically accumulate Spd and Spm in fruits in comparison to fruits from control (556AZ) plants (Mehta et al., 2002). Tomato fruits undergo chilling injury at temperatures below 13°C. The high Spd and Spm tomato together with the control azygous line were utilized to address role(s) of polyamines in chilling-injury signaling. Exposure to chilling temperature (2°C) led to several-fold increase in the Put content in all the lines. Upon re-warming of the fruits at 20°C, the levels of Spd and Spm increased further in the fruit of the two transgenic lines, the higher levels remaining stable for 15 days after re-warming as compared to the fruit from the control line. Profiling their steady state proteins before and after re-warming highlighted a protein of ∼14 kD. Using proteomics approach, protein sequencing and immunoblotting, the ∼14-kD protein was identified as the pathogenesis related protein 1b1 (PR1b1). The PR1b1 protein accumulated transiently in the control fruit whose level was barely detectable at d 15 post-warming while in the fruit from both the 556HO and 579HO transgenic lines PR1b1 abundance increased and remained stable till d 15 post warming. PR1b1 gene transcripts were found low in the control fruit with a visible accumulation only on d 15 post warming; however, in both the transgenic lines it accumulated and increased soon after rewarming being several-fold higher on day 2 while in 556HO line this increase continued until d 6 than the control fruit. The chilling-induced increase in PR1b1 protein seems independent of ethylene and methyl jasmonate signaling but may be linked to salicylic acid. We propose that polyamine-mediated sustained accumulation of PR1b1 protein in post-warmed chilled tomato fruit is a pre-emptive cold stress response and possibly a defense response mechanism related to Cold Stress-Induced Disease Resistance (SIDR) phenomenon.

Introduction: Metabolomics provides a view of endogenous metabolic patterns not only during plant growth, development and senescence but also in response to genetic events, environment and disease. The effects of the field environment on plant hormone-specific metabolite profiles are largely unknown. Few studies have analyzed useful phenotypes generated by introducing single or multiple gene events alongside the non-engineered wild type control at field scale to determine the robustness of the genetic trait and its modulation in the metabolome as a function of specific agroecosystem environments., Objectives: We evaluated the influence of genetic background (high polyamine lines; low methyl jasmonate line; low ethylene line; and isogenic genotypes carrying double transgenic events) and environments (hairy vetch, rye, plastic black mulch and bare soil mulching systems) on the metabolomic profile of isogenic reverse genetic mutations and selected mulch based cropping systems in tomato fruit. Net photosynthesis and fruit yield were also determined., Methods: NMR spectroscopy was used for quantifying metabolites that are central to primary metabolism. We analyzed both the first moment (means) of metabolic response to genotypes and agroecosystems by traditional univariate/multivariate methods, and the second moment (covariances) of responses by creating networks that depicted changes in correlations of paired metabolites. This particular approach is novel and was necessary because our experimental material yielded highly variable metabolic responses that could not be easily understood using the traditional analytical approaches for first moment statistics., Results: High endogenous spermidine and spermine content exhibited strong effects on amino acids, Krebs cycle intermediates and energy molecules (ADP + ATP) in ripening fruits of plants grown under different agroecosystem environments. The metabolic response to high polyamine genotypes was similar to the response to hairy vetch cover crop mulch; supported by the pattern of changes in correlation between metabolites. Changes in primary metabolites of genotypes mutated for the deficiency of ethylene or methyl jasmonate were unique under all growth conditions and opposite of high polyamine genotype results. The high polyamine trait was found to dominate the low ethylene and low jasmonate mutations under field conditions. For several metabolites low ethylene and low methyl jasmonate genotypes had an inverse relationship. Collectively, these results affirm that interactions between metabolite pathways and growth environments are affected by genotype, and influence the metabolite quality of a crop., Conclusion: This study portrays how metabolite relationships change, both in mean and in correlation, under different genotypic and environmental conditions. Although these networks are surprisingly dynamic, we also find examples of selectively conserved associations.

Biotypes of the broad-leaved wild mustard (Sinapis arvensis L.) found in wheat fields of Aegean and Marmara region of Turkey were characterized and shown to have developed resistance to sulfonylurea (chlorsulfuron), an inhibitor of acetolactate synthase (ALS). DNA sequence analysis of the ALS genes from two such resistant ("R") biotypes, KNF-R1 and KNF-R2, revealed point mutations, CCT (Pro 197) to TCT (Ser 197) in KNF-R1 and CCT (Pro 197) to ACT (Thr 197) in KNF-R2; these substitutions are consistent with the presence of chlorsulfuron-insensitive ALS enzyme activity in the "R" S. arvensis biotypes. An additional phenotype of chlorsulfuron resistance in the Turkish S. arvensis "R" biotypes was revealed in the form of an altered seed dormancy behavior over 4-48 months of dry storage (after-ripening) compared to the susceptible ("S") biotypes. Seeds of the "S" biotypes dry stored for 4 months had a higher initial germination, which sharply decreased with storage time, while the seeds of the "R" biotypes had lower germination after 4-months storage, rising sharply and peaking thereafter by 24 months' of dry storage. The "R" biotype seeds continued to maintain a higher germination percentage even after 48 months of after-ripening. The seed weight of "R" and "S" biotypes after-ripened for 4 months was similar but those after-ripened for 48 months differed, "R" seeds were significantly heavier than those of the "S" seeds. Differential seed germinability between "S" and "R" biotypes was found not a case of differential viability, temperature regimen or non-response to pro-germination hormone GA3. These studies are of relevance to ecological fitness of herbicide-resistant biotypes in terms of seed viability and germination.

Ethylene regulates a myriad physiological and biochemical processes in ripening fruits and is accepted as the ripening hormone for the climacteric fruits. However, its effects on metabolome and resulting fruit quality are not yet fully understood, particularly when some of the ripening-associated biochemical changes are independent of ethylene action. We have generated a homozygous transgenic tomato genotype (2AS-AS) that exhibits reduced ethylene production as a result of impaired expression of 1-aminocyclopropane-1-carboxylate synthase 2 gene by its antisense RNA and had a longer shelf life. Double transgenic hybrid (2AS-AS × 579HO) developed through a genetic cross between 2AS-AS and 579HO (Mehta et al., 2002) lines resulted in significantly higher ethylene production than either the WT or 2AS-AS fruit. To determine the effects of reduced ethylene and introgression of higher polyamines' trait, the metabolic profiles of ripening fruits from WT (556AZ), 2AS-AS, and 2AS-AS × 579HO lines were determined using (1)H-NMR spectroscopy. The levels of Glu, Asp, AMP, Adenosine, Nucl1, and Nucl2 increased during ripening of the WT fruit. The increases in Glu, Asp, and AMP levels were attenuated in 2AS-AS fruit but recovered in the double hybrid with higher ethylene and polyamine levels. The ripening-associated decreases in Ala, Tyr, Val, Ile, Phe, malate, and myo-inositol levels in the 2AS-AS line were not reversed in the double hybrid line suggesting a developmental/ripening regulated accumulation of these metabolites independent of ethylene. Significant increases in the levels of fumarate, formate, choline, Nucl1, and Nucl2 at most stages of ripening fruit were found in the double transgenic line due to introgression with higher-polyamines trait. Taken together these results show that the ripening-associated metabolic changes are both ethylene dependent and independent, and that the fruit metabolome is under the control of multiple regulators, including ethylene and polyamines.

Crop losses due to pathogens are a major threat to global food security. Plants employ a multilayer defense against a pathogen including the use of physical barriers (cell wall), induction of hypersensitive defense response (HR), resistance (R) proteins, and synthesis of antimicrobial peptides (AMPs). Unlike a complex R gene-mediated immunity, AMPs directly target diverse microbial pathogens. Many a times, R-mediated immunity breaks down and plant defense is compromised. Although R-gene dependent pathogen resistance has been well studied, comparatively little is known about the interactions of AMPs with host defense and physiology. AMPs are ubiquitous, low molecular weight peptides that display broad spectrum resistance against bacteria, fungi and viruses. In plants, AMPs are mainly classified into cyclotides, defensins, thionins, lipid transfer proteins, snakins, and hevein-like vicilin-like and knottins. Genetic distance lineages suggest their conservation with minimal effect of speciation events during evolution. AMPs provide durable resistance in plants through a combination of membrane lysis and cellular toxicity of the pathogen. Plant hormones - gibberellins, ethylene, jasmonates, and salicylic acid, are among the physiological regulators that regulate the expression of AMPs. Transgenically produced AMP-plants have become a means showing that AMPs are able to mitigate host defense responses while providing durable resistance against pathogens., (Published by Elsevier Ireland Ltd.)

S-adenosylmethionine (SAM), a major substrate in 1-C metabolism is a common precursor in the biosynthetic pathways of polyamines and ethylene, two important plant growth regulators, which exhibit opposing developmental effects, especially during fruit ripening. However, the flux of various substrates including SAM into the two competing pathways in plants has not yet been characterized. We used radiolabeled (14)C-Arg, (14)C-Orn, L-[U-(14)C]Met, (14)C-SAM and (14)C-Put to quantify flux through these pathways in tomato fruit and evaluate the effects of perturbing these pathways via transgenic expression of a yeast SAM decarboxylase (ySAMDC) gene using the fruit ripening-specific promoter E8. We show that polyamines in tomato fruit are synthesized both from Arg and Orn; however, the relative contribution of Orn pathway declines in the later stages of ripening. Expression of ySAMDC reversed the ripening associated decline in spermidine (Spd) and spermine (Spm) levels observed in the azygous control fruit. About 2- to 3-fold higher levels of labeled-Spd in transgenic fruit (556HO and 579HO lines) expressing ySAMDC confirmed the enzymatic function of the introduced gene. The incorporation of L-[U-(14)C]Met into Spd, Spm, ethylene and 1-aminocyclopropane-1-carboxylic acid (ACC) was used to determine Met-flux into these metabolites. The incorporation of (14)C-Met into Spd/Spm declined during ripening of the control azygous fruit but this was reversed in fruits expressing ySAMDC. However, incorporation of (14)C-Met into ethylene or ACC during ripening was not altered by the expression of ySAMDC in the fruit. Taken together these results show that: (1) There is an inverse relationship between the production of higher polyamines and ethylene during fruit ripening, (2) the inverse relationship between higher polyamines and ethylene is modulated by ySAMDC expression in that the decline in Spd/Spm during fruit ripening can be reversed without significantly altering ethylene biosynthesis, and (3) cellular flux of SAM in plants is homeostatically regulated based on its demand for competing pathways.

Antimicrobial cationic peptides (AMPs) are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s) and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani) and abiotic stressors (dark-induced senescence, wounding and temperature stress). msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR) and reactive oxygen species (ROS) responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s) of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

Space missions have enabled testing how microorganisms, animals and plants respond to extra-terrestrial, complex and hazardous environment in space. Photosynthetic organisms are thought to be relatively more prone to microgravity, weak magnetic field and cosmic radiation because oxygenic photosynthesis is intimately associated with capture and conversion of light energy into chemical energy, a process that has adapted to relatively less complex and contained environment on Earth. To study the direct effect of the space environment on the fundamental process of photosynthesis, we sent into low Earth orbit space engineered and mutated strains of the unicellular green alga, Chlamydomonas reinhardtii, which has been widely used as a model of photosynthetic organisms. The algal mutants contained specific amino acid substitutions in the functionally important regions of the pivotal Photosystem II (PSII) reaction centre D1 protein near the QB binding pocket and in the environment surrounding Tyr-161 (YZ) electron acceptor of the oxygen-evolving complex. Using real-time measurements of PSII photochemistry, here we show that during the space flight while the control strain and two D1 mutants (A250L and V160A) were inefficient in carrying out PSII activity, two other D1 mutants, I163N and A251C, performed efficient photosynthesis, and actively re-grew upon return to Earth. Mimicking the neutron irradiation component of cosmic rays on Earth yielded similar results. Experiments with I163N and A251C D1 mutants performed on ground showed that they are better able to modulate PSII excitation pressure and have higher capacity to reoxidize the QA (-) state of the primary electron acceptor. These results highlight the contribution of D1 conformation in relation to photosynthesis and oxygen production in space.

Excessive use of nitrogen (N) in crop production has impacted ecosystems by contaminating soil and water. Management of N in agriculture is therefore of global concern. Sustainable agriculture systems that use leguminous cover crops such as hairy vetch (Vicia villosa Roth) to fix N and enrich soil organic matter by fixing carbon provide an alternative strategy. N signalling pathways were found associated with delayed leaf senescence and disease tolerance of hairy vetch-grown tomatoes. To test whether N in hairy vetch is the only contributing factor leading to these phenotypes, we designed a pot experiment in the field to analyse growth and gene expression in tomatoes, one set with soil overwintered without a cover crop (bare soil) and the other with soil overwintered with a hairy vetch cover crop including the vetch residue on the soil surface. Additionally, supplementary N fertiliser was also provided to aid distinguishing tomato responses to vetch from those to inorganic N. Tomato fruit yield, plant biomass and photosynthesis were higher in plants grown in vetch than bare soil. Tomato growth and photosynthesis metrics exhibited a parabolic response to inorganic N in bare soil, suggesting the potential for N toxicity in pots with the highest N rate. Vetch-grown tomato plants mitigated these effects and maintained elevated photosynthetic rates at high inorganic N levels. Vetch-grown plants also mitigated a decline in expression of several genes regulating nitrogen and carbon metabolism and upregulated the defence-related gene, osmotin, relative to plants grown in bare soil. Thus, some of the positive responses of tomatoes to a hairy vetch cover crop observed in the field seem mediated by physiological cues other than the additional N provided by the vetch cover crop.

Physical clustering of genes has been shown in plants; however, little is known about gene clusters that have different functions, particularly those expressed in the tomato fruit. A class I 17.6 small heat shock protein (Sl17.6 shsp) gene was cloned and used as a probe to screen a tomato (Solanum lycopersicum) genomic library. An 8.3-kb genomic fragment was isolated and its DNA sequence determined. Analysis of the genomic fragment identified intronless open reading frames of three class I shsp genes (Sl17.6, Sl20.0, and Sl20.1), the Sl17.6 gene flanked by Sl20.1 and Sl20.0, with complete 5' and 3' UTRs. Upstream of the Sl20.0 shsp, and within the shsp gene cluster, resides a box C/D snoRNA cluster made of SlsnoR12.1 and SlU24a. Characteristic C and D, and C' and D', boxes are conserved in SlsnoR12.1 and SlU24a while the upstream flanking region of SlsnoR12.1 carries TATA box 1, homol-E and homol-D box-like cis sequences, TM6 promoter, and an uncharacterized tomato EST. Molecular phylogenetic analysis revealed that this particular arrangement of shsps is conserved in tomato genome but is distinct from other species. The intronless genomic sequence is decorated with cis elements previously shown to be responsive to cues from plant hormones, dehydration, cold, heat, and MYC/MYB and WRKY71 transcription factors. Chromosomal mapping localized the tomato genomic sequence on the short arm of chromosome 6 in the introgression line (IL) 6-3. Quantitative polymerase chain reaction analysis of gene cluster members revealed differential expression during ripening of tomato fruit, and relatively different abundances in other plant parts.

Transgenic tomato (Solanum lycopersicum) lines overexpressing yeast spermidine synthase (ySpdSyn), an enzyme involved in polyamine (PA) biosynthesis, were developed. These transgenic lines accumulate higher levels of spermidine (Spd) than the wild-type plants and were examined for responses to the fungal necrotrophs Botrytis cinerea and Alternaria solani, bacterial pathogen Pseudomonas syringae pv tomato DC3000, and larvae of the chewing insect tobacco hornworm (Manduca sexta). The Spd-accumulating transgenic tomato lines were more susceptible to B. cinerea than the wild-type plants; however, responses to A. solani, P. syringae, or M. sexta were similar to the wild-type plants. Exogenous application of ethylene precursors, S-adenosyl-Met and 1-aminocyclopropane-1-carboxylic acid, or PA biosynthesis inhibitors reversed the response of the transgenic plants to B. cinerea. The increased susceptibility of the ySpdSyn transgenic tomato to B. cinerea was associated with down-regulation of gene transcripts involved in ethylene biosynthesis and signaling. These data suggest that PA-mediated susceptibility to B. cinerea is linked to interference with the functions of ethylene in plant defense.

Exogenous treatment with jasmonates (JA) has been shown to reduce the levels of polyamines in many plants. But the role of endogenous JA on polyamine biosynthesis or other cellular metabolites has thus far remained uninvestigated. We developed transgenic tomato (Solanum lycopersicum L.) having severely reduced methyl JA levels by silencing a fruit ripening-associated lipoxygenase (LOX), SlLoxB, using a truncated LOX gene under the control of the constitutive CaMV35S promoter. The LOX suppressed and MeJA-deficient fruits had lowered polyamine levels. Thus, these transgenic fruits were used as a plant model to evaluate the effects of reduced endogenous MeJA on cellular metabolites in ripening tomato fruits using NMR spectroscopy. During on-shelf ripening, transgenic fruits were significantly reduced in the content of 19 out of 30 metabolites examined, including Ile, Val, Ala, Thr, Asn Tyr, Glu, Gln, His, Phe, Trp, GABA, citrate, succinate, myo-inositol, unidentified compound B, nucleic acid compound Nucl1, choline, and trigonelline as compared to the wild-type azygous counterparts. A significant increase in β-glucose levels in transgenic fruits was observed at the pink stage. The transgenic fruits were equivalent to the wild type in lycopene level and chlorophyll degradation rates. Taken together, these results show that intracellular MeJA significantly regulates overall primary metabolism, especially aminome (amino acids and polyamines) of ripening fruits.

Polyamines (PAs) are ubiquitous, polycationic biogenic amines that are implicated in many biological processes, including plant growth and development, but their precise roles remain to be determined. Most of the previous studies have involved three biogenic amines: putrescine (Put), spermidine (Spd) and spermine (Spm), and their derivatives. We have expressed a yeast spermidine synthase (ySpdSyn) gene under constitutive (CaMV35S) and fruit-ripening specific (E8) promoters in Solanum lycopersicum (tomato), and determined alterations in tomato vegetative and fruit physiology in transformed lines compared with the control. Constitutive expression of ySpdSyn enhanced intracellular levels of Spd in the leaf, and transiently during fruit development, whereas E8-ySpdSyn expression led to Spd accumulation early and transiently during fruit ripening. The ySpdSyn transgenic fruits had a longer shelf life, reduced shriveling and delayed decay symptom development in comparison with the wild-type (WT) fruits. An increase in shelf life of ySpdSyn transgenic fruits was not facilitated by changes in the rate of water loss or ethylene evolution. Additionally, the expression of several cell wall and membrane degradation-related genes in ySpdSyn transgenic fruits was not correlated with an extension of shelf life, indicating that the Spd-mediated increase in fruit shelf life is independent of the above factors. Crop maturity, indicated by the percentage of ripening fruits on the vine, was delayed in a CaMV35S-ySpdSyn genotype, with fruits accumulating higher levels of the antioxidant lycopene. Notably, whole-plant senescence in the transgenic plants was also delayed compared with WT plants. Together, these results provide evidence for a role of PAs, particularly Spd, in increasing fruit shelf life, probably by reducing post-harvest senescence and decay., (No claim to original US government works. Journal compilation © 2010 Blackwell Publishing Ltd.)

Biogenic amines putrescine, spermidine and spermine are ubiquitous in nature and have interested researchers because they are essential for cell division and viability, and due to a large body of their pharmacological effects on growth and development in most living cells. The genes and enzymes involved in their biosynthetic pathways are now established and characterized. In recent years, molecular aspects of polyamine action have also begun to emerge. Our model is the ripening tomato fruit in which processes of cell division, cell expansion and cell growth have ceased, and yet the cells are responsive at biochemical and molecular levels to genetically manipulated concentrations of putrescine (Put), spermidine (Spd) and spermine (Spm). Thus, transcriptome, limited protein profiling, and metabolome studies of transgenic tomato fruit have yielded significant new information on cellular processes impacted by polyamine manipulation. We have used these datasets to determine the linear correlation coefficients between the endogenous levels of Put, Spd and Spm with several parameters. Results of our analysis presented here show that effects of the diamine Put generally contrast those with polyamines Spd and Spm, emphasizing that individual biogenic amines should be considered to have defined action in plant biology and that they differentially affect growth and development. A multiple function model of polyamine action is discussed to explain the role of polyamines in most organisms, in general, and ripening fruit, in particular., (Published by Elsevier Masson SAS.)

Tomato (Solanum lycopersicum) fruit is a model to study molecular basis of fleshy fruit development and ripening. We profiled gene expression during fruit development (immature green and mature green fruit) and ripening (breaker stage onwards) program to obtain a global perspective of genes whose expression is modulated at each stage of fruit development and ripening. A custom made cDNA macroarray containing cDNAs representing various metabolic pathways, defense, signaling, transcription, transport, cell structure and cell wall related functions was developed and used to quantify changes in the abundance of different transcripts. About 34 % of 1066 unique expressed sequence tags (ESTs) printed on the macroarray were differentially expressed during tomato fruit ripening. Out of these, 25 % genes classify under metabolism and protein biosynthesis/degradation related processes, while a significant proportion represented stress-responsive genes and about 44 % represented genes with unknown functions. RNA gel blot analysis validated changes in a few representative genes. Although the mature green fruit was found transcriptionally quiescent, the K-means cluster analysis highlighted coordinated up or down regulation of genes during progressive ripening; emphasizing that ripening is a transcriptionally active process. Many stress-related genes were found up-regulated, suggesting their role in the fruit ripening program.

Distribution of biogenic amines-the diamine putrescine (Put), triamine spermidine (Spd), and tetraamine spermine (Spm)-differs between species with Put and Spd being particularly abundant and Spm the least abundant in plant cells. These amines are important for cell viability and their intracellular levels are tightly regulated, which have made it difficult to characterize individual effects of Put, Spd and Spm on plant growth and developmental processes. The recent transgenic intervention and mutational genetics have made it possible to stably alter levels of naturally occurring polyamines and study their biological effects. We bring together an analysis of certain metabolic changes, particularly in amino acids, to infer the responsive regulation brought about by increased diamine or polyamine levels in actively growing poplar cell cultures (transformed with mouse ornithine decarboxylase gene to accumulate high Put levels) and ripening tomato pericarp (transformed with yeast S-adenosylmethionine decarboxylase gene to accumulate high Spd and Spm levels at the cost of Put). Our analysis indicates that increased Put has little effect on increasing the levels of Spd and Spm, while Spd and Spm levels are inter-dependent. Further, Put levels were positively associated with Ala (alpha and beta), Ile and GABA and negatively correlated with Gln and Glu in both actively growing poplar cell cultures and non-dividing tomato pericarp tissue. Most amino acids showed positive correlations with Spd and Spm levels in actively growing cells. Collectively these results suggest that Put is a negative regulator while Spd-Spm are positive regulators of cellular amino acid metabolism.

The 1H-NMR methodology used in the study of genetically modified (GM) foods is discussed. Transgenic lettuce (Lactuca sativa cv "Luxor") over-expressing the ArabidopsisKNAT1 gene is presented as a case study. Twenty-two water-soluble metabolites (amino acids, organic acids, sugars) present in leaves of conventional and GM lettuce were monitored by NMR and quantified at two developmental stages. The NMR spectra did not reveal any difference in metabolite composition between the GM lettuce and the wild type counterpart. Statistical analyses of metabolite variables highlighted metabolism variation as a function of leaf development as well as the transgene. A main effect of the transgene was in altering sugar metabolism.