The iron–sulfur cluster biosynthesis protein SUFB is required for chlorophyll synthesis,but not phytochrome signaling

Proteins that contain iron–sulfur (Fe–S) clusters play pivotal roles in various metabolic processes such as photosynthesis and redox metabolism. Among the proteins involved in the biosynthesis of Fe–S clusters in plants, the SUFB subunit of the SUFBCD complex appears to be unique because SUFB has been reported to be involved in chlorophyll metabolism and phytochrome‐mediated signaling. To gain insights into the function of the SUFB protein, we analyzed the phenotypes of two SUFB mutants, laf6 and hmc1, and RNA interference (RNAi) lines with reduced SUFB expression. When grown in the light, the laf6 and hmc1 mutants and the SUFB RNAi lines accumulated higher levels of the chlorophyll biosynthesis intermediate Mg‐protoporphyrin IX monomethylester (Mg‐proto MME), consistent with the impairment of Mg‐proto MME cyclase activity. Both SUFC‐ and SUFD‐deficient RNAi lines accumulated the same intermediate, suggesting that inhibition of Fe‐S cluster synthesis is the primary cause of this impairment. Dark‐grown laf6 seedlings also showed an increase in protoporphyrin IX (Proto IX), Mg‐proto, Mg‐proto MME and 3,8‐divinyl protochlorophyllide a (DV‐Pchlide) levels, but this was not observed in hmc1 or the SUFB RNAi lines, nor was it complemented by SUFB overexpression. In addition, the long hypocotyl in far‐red light phenotype of the laf6 mutant could not be rescued by SUFB overexpression and segregated from the pale‐green SUFB‐deficient phenotype, indicating it is not caused by mutation at the SUFB locus. These results demonstrate that biosynthesis of Fe–S clusters is important for chlorophyll biosynthesis, but that the laf6 phenotype is not due to a SUFB mutation.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Bacillithiol has a role in Fe–S cluster biogenesis in Staphylococcus aureus

Staphylococcus aureus does not produce the low‐molecular‐weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH‐deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron–sulfur (Fe–S) cluster‐dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH‐deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe–S cluster biogenesis. The phenotypes of the BSH‐deficient strains were exacerbated in strains lacking the Fe–S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe–S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe–S clusters to apo‐aconitase, verifying that it serves as an Fe–S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe–S clusters to apo‐proteins in S. aureus.     

Overexpression of BUNDLE SHEATH DEFECTIVE 2 improves the efficiency of photosynthesis and growth in Arabidopsis

Bundle Sheath Defective 2, BSD2, is a stroma‐targeted protein initially identified as a factor required for the biogenesis of ribulose 1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) in maize. Plants and algae universally have a homologous gene for BSD2 and its deficiency causes a RuBisCO‐less phenotype. As RuBisCO can be the rate‐limiting step in CO₂ assimilation, the overexpression of BSD2 might improve photosynthesis and productivity through the accumulation of RuBisCO. To examine this hypothesis, we produced BSD2 overexpression lines in Arabidopsis. Compared with wild type, the BSD2 overexpression lines BSD2ox‐2 and BSD2ox‐3 expressed 4.8‐fold and 8.8‐fold higher BSD2 mRNA, respectively, whereas the empty‐vector (EV) harbouring plants had a comparable expression level. The overexpression lines showed a significantly higher CO₂ assimilation rate per available CO₂ and productivity than EV plants. The maximum carboxylation rate per total catalytic site was accelerated in the overexpression lines, while the number of total catalytic sites and RuBisCO content were unaffected. We then isolated recombinant BSD2 (rBSD2) from E. coli and found that rBSD2 reduces disulfide bonds using reductants present in vivo, for example glutathione, and that rBSD2 has the ability to reactivate RuBisCO that has been inactivated by oxidants. Furthermore, 15% of RuBisCO freshly isolated from leaves of EV was oxidatively inactivated, as compared with 0% in BSD2‐overexpression lines, suggesting that the overexpression of BSD2 maintains RuBisCO to be in the reduced active form in vivo. Our results demonstrated that the overexpression of BSD2 improves photosynthetic efficiency in Arabidopsis and we conclude that it is involved in mediating RuBisCO activation.     

Involvement of thioredoxin y2 in the preservation of leaf methionine sulfoxide reductase capacity and growth under high light

Methionine (Met) in proteins can be oxidized to two diastereoisomers of methionine sulfoxide, Met‐S‐O and Met‐R‐O, which are reduced back to Met by two types of methionine sulfoxide reductases (MSRs), A and B, respectively. MSRs are generally supplied with reducing power by thioredoxins. Plants are characterized by a large number of thioredoxin isoforms, but those providing electrons to MSRs in vivo are not known. Three MSR isoforms, MSRA4, MSRB1 and MSRB2, are present in Arabidopsis thaliana chloroplasts. Under conditions of high light and long photoperiod, plants knockdown for each plastidial MSR type or for both display reduced growth. In contrast, overexpression of plastidial MSRBs is not associated with beneficial effects in terms of growth under high light. To identify the physiological reductants for plastidial MSRs, we analyzed a series of mutants deficient for thioredoxins f, m, x or y. We show that mutant lines lacking both thioredoxins y1 and y2 or only thioredoxin y2 specifically display a significantly reduced leaf MSR capacity (–25%) and growth characteristics under high light, related to those of plants lacking plastidial MSRs. We propose that thioredoxin y2 plays a physiological function in protein repair mechanisms as an electron donor to plastidial MSRs in photosynthetic organs.     

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase,confers salinity and cold tolerance in tobacco

Calcineurin B‐like protein‐interacting protein kinases (CIPKs) are components of Ca²⁺ signaling in responses to abiotic stresses. In this work, the full‐length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT‐PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H₂O₂. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H₂O₂ and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na⁺ content in the transgenic lines under salt stress. Higher expression of stress‐related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress‐responsive gene in plants.     

Chlorophyll accumulation and breakdown in light-grown barley transferred to darkness: effect of seedling age

Diurnally grown barley (Hordeum vulgare L. cv. Clipper) seedlings of various ages (3–4, 5–6 and 10–11-days-old) were transferred to darkness for 17 h and changes in leaf fresh weight, chlorophyll a, chlorophyll b and protochlorophyllide measured. The results were consistent with previous evidence of a light-independent chlorophyll biosynthetic pathway in light-grown barley. There was a net gain in chlorophyll (μg leaf^-1) in 5–6- and 10–11-day-old plants after 17 h dark treatment. The amounts of chlorophyll that accumulated were similar (5.9 and 4.3 μg Chl leaf^-1), despite a twofold difference in leaf size at T₀. The rate of leaf expansion in 5–6-day-old plants greatly exceeded the rate of chlorophyll accumulation and leaves were noticeably paler after dark treatment i.e. there was a reduction in chlorophyll concentration (μg g fresh weight^-1) in spite of an increase in chlorophyll content (μg leaf^-1). The ability of light-grown barley to accumulate chlorophyll in darkness was a function of seedling age. Very young seedlings (3–4-day-old) generally lost chlorophyll in darkness. The decrease in chlorophyll per leaf resulted mainly from loss of chlorophyll b. Preferential loss of chlorophyll b resulted in dramatic increases in the chlorophyll a:b ratio. Since 3–4-day-old seedlings (1) accumulated 5-aminolevulinic acid in the presence of levulinic acid at a rate comparable to older seedlings, and (2) converted exogenous 5-aminolevulinic acid to chlorophyll in the absence of light, it is unlikely that failure of the youngest plants to accumulate chlorophyll in darkness was due to blocks at these steps in the pathway. Net loss of chlorophyll (μg leaf^-1) in 3–4-day-old seedlings in darkness was eliminated by the addition of chloramphenicol, which occasionally produced a small, but significant, gain in total chlorophyll. These results imply that chlorophyll degradation in young barley leaves is strongly influenced by the chloroplast genome, and is a major factor influencing changes in chlorophyll levels in darkness. The present findings are consistent with the suggestion that the failure of 3–4-day-old barley seedlings to accumulate chlorophyll in darkness may be due to chlorophyll turnover in which the rate of degradation exceeds the rate of synthesis.     

Overexpression of AtABCG36 improves drought and salt stress resistance in Arabidopsis

Drought and salt are major abiotic stresses that adversely affect crop productivity. Thus, identification of factors that confer resistance to these stresses would pave way to increasing agricultural productivity. When grown on soil in green house longer than 5 weeks, transgenic Arabidopsis plants that overexpress an ATP‐binding cassette (ABC) transporter, AtABCG36/AtPDR8, produced higher shoot biomass and less chlorotic leaves than the wild‐type. We investigated whether the improved growth of AtABCG36‐overexpressing plants was due to their improved resistance to abiotic stresses, and found that AtABCG36‐overexpressing plants were more resistant to drought and salt stress and grew to higher shoot fresh weight (FW) than the wild‐type. On the contrary, T‐DNA insertional knockout lines were more sensitive to drought stress than wild‐type and were reduced in shoot FW. To understand the mechanism of enhanced salt and drought resistance of the AtABCG36 overexpressing plants, we measured sodium contents and found that AtABCG36 overexpressing plants were lower in sodium content than the wild‐type. Our data suggest that AtABCG36 contributes to drought and salt resistance in Arabidopsis by a mechanism that includes reduction of sodium content in plants.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> FtnA acts as an iron buffer for re-assembly of iron–sulfur clusters in response to hydrogen peroxide stress

Iron–sulfur clusters are one of the most ubiquitous redox centers in biology. Ironically, iron-sulfur clusters are highly sensitive to reactive oxygen species. Disruption of iron-sulfur clusters will not only change the activity of proteins that host iron–sulfur clusters, the iron released from the disrupted iron–sulfur clusters will further promote the production of deleterious hydroxyl free radicals via the Fenton reaction. Here, we report that ferritin A (FtnA), a major iron-storage protein in Escherichia coli, is able to scavenge the iron released from the disrupted iron–sulfur clusters and alleviates the production of hydroxyl free radicals. Furthermore, we find that the iron stored in FtnA can be retrieved by an iron chaperon IscA for the re-assembly of the iron–sulfur cluster in a proposed scaffold IscU in the presence of the thioredoxin reductase system which emulates normal intracellular redox potential. The results suggest that E. coli FtnA may act as an iron buffer to sequester the iron released from the disrupted iron–sulfur clusters under oxidative stress conditions and to facilitate the re-assembly of the disrupted iron–sulfur clusters under normal physiological conditions.     

Genome wide identification of the trihelix transcription factors and overexpression of Gh_A05G2067 (GT‐2), a novel gene contributing to increased drought and salt stresses tolerance in cotton

We identified 102, 51 and 51 proteins encoded by the trihelix genes in Gossypium hirsutum, Gossypium arboreum and Gossypium raimondii, respectively. RNA sequence data and real‐time quantitative polymerase chain reaction analysis showed that Gh_A05G2067 (GT‐2) was highly upregulated under drought and salt stress conditions. Transient expression of GT‐2‐green fluorescent protein fusion protein in protoplast showed that GT‐2 was localized in the nucleus. The overexpression of GT‐2 conferred an enhanced drought tolerance to cotton, with lower malondialdehyde, hydrogen peroxide contents and higher reactive oxygen scavenging enzyme activities. Moreover, chlorophyll content, relative leaf water content (RLWC), excised leaf water loss (ELWL) and cell membrane stability (CMS) were relatively stable in the GT‐2‐overexpressed lines compared to wild‐type (WT). Similarly, stress‐responsive genes RD29A, SOS1, ABF4 and CBL1 were highly upregulated in the GT‐2‐overexpressed lines but were significantly downregulated in WT. In addition, the GT‐2‐silenced cotton plants exhibited a high level of oxidation injury, due to high levels of oxidant enzymes, in addition to negative effects on CMS, ELWL, RLWC and chlorophyll content. These results mark the foundation for future exploration of the trihelix genes in cotton, with an aim of developing more resilient, versatile and highly tolerant cotton genotypes.     

High temperatures change the perspective: Integrating hormonal responses in citrus plants under co‐occurring abiotic stress conditions

Plants growing in the field are subjected to multiple stress factors acting simultaneously. Abnormally high temperatures are expected to affect wild plants and crops in the next years due to global warming. In this work, we have studied physiological, hormonal and molecular responses of the citrus rootstock, Carrizo citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) subjected to wounding or high salinity occurring individually or in combination with heat stress. According to our results, combination of high salinity and heat stress aggravated the negative effects of salt intoxication in Carrizo. The high transpiration rate caused by high temperatures counteracted physiological responses of plants to salt stress and increased Cl^? intake in leaves. In addition, 12‐oxo‐phytodienoic acid accumulated specifically under combination of wounding and heat stress, whereas at low temperatures, wounded plants accumulated jasmonic acid (JA) and JA‐isoleucine (JA‐Ile). Moreover, an antagonism between salicylic acid (SA) and JA was observed, and wounded plants subjected to high temperatures did not accumulate JA nor JA‐Ile whereas SA levels increased (via isochorismate synthase biosynthetic pathway). Wounded plants did not accumulate abscisic acid (ABA) but its catabolite phaseic acid. This could act as a signal for the upregulation of (ABA)‐RESPONSIVE ELEMENT (ABRE)‐BINDING TRANSCRIPTION FACTOR 2 (CsAREB2) and RESPONSIVE TO DISSECATION 22 (CsRD22) in an ABA‐independent way. This work uncovers some mechanisms that explain Carrizo citrange tolerance to high temperatures together with different hormonal signals in response to specific stresses. It is suggested that co‐occurring abiotic stress conditions can modify (either enhance or reduce) the hormonal response to modulate specific responses.     

Amino acid transporter mutants of Arabidopsis provides evidence that a non‐mycorrhizal plant acquires organic nitrogen from agricultural soil

Although organic nitrogen (N) compounds are ubiquitous in soil solutions, their potential role in plant N nutrition has been questioned. We performed a range of experiments on Arabidopsis thaliana genetically modified to enhance or reduce root uptake of amino acids. Plants lacking expression of the Lysine Histidine Transporter 1 (LHT1) displayed significantly lower contents of ¹³C and ¹⁵N label and of U‐¹³C₅,¹⁵N₂ L‐glutamine, as determined by liquid chromatography–mass spectrometry when growing in pots and supplied with dually labelled L‐glutamine compared to wild type plants and LHT1‐overexpressing plants. Slopes of regressions between accumulation of ¹³C‐labelled carbon and ¹⁵N‐labelled N were higher for LHT1‐overexpressing plants than wild type plants, while plants lacking expression of LHT1 did not display a significant regression between the two isotopes. Uptake of labelled organic N from soil tallied with that of labelled ammonium for wild type plants and LHT1‐overexpressing plants but was significantly lower for plants lacking expression of LHT1. When grown on agricultural soil plants lacking expression of LHT1 had the lowest, and plants overexpressing LHT1 the highest C/N ratios and natural δ¹⁵N abundance suggesting their dependence on different N pools. Our data show that LHT1 expression is crucial for plant uptake of organic N from soil.     

Transgenic banana plants overexpressing a native plasma membrane aquaporin MusaPIP1;2 display high tolerance levels to different abiotic stresses

Water transport across cellular membranes is regulated by a family of water channel proteins known as aquaporins (AQPs). As most abiotic stresses like suboptimal temperatures, drought or salinity result in cellular dehydration, it is imperative to study the cause–effect relationship between AQPs and the cellular consequences of abiotic stress stimuli. Although plant cells have a high isoform diversity of AQPs, the individual and integrated roles of individual AQPs in optimal and suboptimal physiological conditions remain unclear. Herein, we have identified a plasma membrane intrinsic protein gene (MusaPIP1;2) from banana and characterized it by overexpression in transgenic banana plants. Cellular localization assay performed using MusaPIP1;2::GFP fusion protein indicated that MusaPIP1;2 translocated to plasma membrane in transformed banana cells. Transgenic banana plants overexpressing MusaPIP1;2 constitutively displayed better abiotic stress survival characteristics. The transgenic lines had lower malondialdehyde levels, elevated proline and relative water content and higher photosynthetic efficiency as compared to equivalent controls under different abiotic stress conditions. Greenhouse‐maintained hardened transgenic plants showed faster recovery towards normal growth and development after cessation of abiotic stress stimuli, thereby underlining the importance of these plants in actual environmental conditions wherein the stress stimuli is often transient but severe. Further, transgenic plants where the overexpression of MusaPIP1;2 was made conditional by tagging it with a stress‐inducible native dehydrin promoter also showed similar stress tolerance characteristics in in vitro and in vivo assays. Plants developed in this study could potentially enable banana cultivation in areas where adverse environmental conditions hitherto preclude commercial banana cultivation.