Medicago truncatula Yellow Stripe-Like7 encodes a peptide transporter participating in symbiotic nitrogen fixation.

Yellow Stripe-Like (YSL) proteins are a family of plant transporters that are typically involved in transition metal homeostasis. Three of the four YSL clades (I, II and IV) transport metals complexed with the non-proteinogenic amino acid nicotianamine or its derivatives. No such capability has been shown for any member of clade III, but the link between these YSLs and metal homeostasis could be masked by functional redundancy. We studied the role of the clade III YSL protein MtSYL7 in Medicago truncatula nodules. MtYSL7, which encodes a plasma membrane-bound protein, is mainly expressed in the pericycle and cortex cells of the root nodules. Yeast complementation assays revealed that MtSYL7 can transport short peptides. M. truncatula transposon insertion mutants with decreased expression of MtYSL7 had lower nitrogen fixation rates and showed reduced plant growth whether grown in symbiosis with rhizobia or not. YSL7 mutants accumulated more copper and iron in the nodules, which is likely to result from the increased expression of iron uptake and delivery genes in roots. Taken together, these data suggest that MtYSL7 plays an important role in the transition metal homeostasis of nodules and symbiotic nitrogen fixation.

Iron-Nicotianamine Transporters Are Required for Proper Long Distance Iron Signaling.

The mechanisms of root iron uptake and the transcriptional networks that control root-level regulation of iron uptake have been well studied, but the mechanisms by which shoots signal iron status to the roots remain opaque. Here, we characterize an Arabidopsis (Arabidopsis thaliana) double mutant, yellow stripe1-like yellow stripe3-like (ysl1ysl3), which has lost the ability to properly regulate iron deficiency-influenced gene expression in both roots and shoots. In spite of markedly low tissue levels of iron, the double mutant does not up- and down-regulate iron deficiency-induced and -repressed genes. We have used grafting experiments to show that wild-type roots grafted to ysl1ysl3 shoots do not initiate iron deficiency-induced gene expression, indicating that the ysl1ysl3 shoots fail to send an appropriate long-distance signal of shoot iron status to the roots. We present a model to explain how impaired iron localization in leaf veins results in incorrect signals of iron sufficiency being sent to roots and affecting gene expression there. Improved understanding of the mechanism of long-distance iron signaling will allow improved strategies for the engineering of staple crops to accumulate additional bioavailable iron in edible parts, thus improving the iron nutrition of the billions of people worldwide whose inadequate diet causes iron deficiency anemia.

Methyl jasmonate represses growth and affects cell cycle progression in cultured Taxus cells.

KEY MESSAGE: Methyl jasmonate elicitation of Taxus cultures enhances paclitaxel accumulation, but represses growth by inhibition of cell cycle progression. Growth repression is evident both at the culture level and transcriptional level. Methyl jasmonate (MeJA) elicitation is an effective strategy to induce and enhance synthesis of the anticancer agent paclitaxel (Taxol(®)) in Taxus cell suspension cultures; however, concurrent decreases in growth are often observed, which is problematic for large-scale bioprocessing. Here, increased accumulation of paclitaxel in Taxus cuspidata suspension cultures with MeJA elicitation was accompanied by a concomitant decrease in cell growth, evident within the first 3 days post-elicitation. Both MeJA-elicited and mock-elicited cultures exhibited similar viability with no apoptosis up to day 16 and day 24 of the cell culture period, respectively, suggesting that growth repression is not attributable to cell death. Flow cytometric analyses demonstrated that MeJA perturbed cell cycle progression of asynchronously dividing Taxus cells. MeJA slowed down cell cycle progression, impaired the G1/S transition as observed by an increase in G0/G1 phase cells, and decreased the number of actively dividing cells. Through a combination of deep sequencing and gene expression analyses, the expression status of Taxus cell cycle-associated genes correlated with observations at the culture level. Results from this study provide valuable insight into the mechanisms governing MeJA perception and subsequent events leading to repression of Taxus cell growth.

The Small RNA Component of Arabidopsis thaliana Phloem Sap and Its Response to Iron Deficiency.

In order to discover sRNA that might function during iron deficiency stress, RNA was prepared from phloem exudates of Arabidopsis thaliana, and used for RNA-seq. Bioanalyzer results indicate that abundant RNA from phloem is small in size-less than 200 nt. Moreover, typical rRNA bands were not observed. Sequencing of eight independent phloem RNA samples indicated that tRNA-derived fragments, specifically 5' tRFs and 5' tRNA halves, are highly abundant in phloem sap, comprising about 46% of all reads. In addition, a set of miRNAs that are present in phloem sap was defined, and several miRNAs and sRNAs were identified that are differentially expressed during iron deficiency.

Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells.

BACKGROUND: Taxol(®) (paclitaxel) promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ) elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH) to identify genes involved in global pathway control. RESULTS: Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day) to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO) analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. CONCLUSIONS: This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

Genetic and biochemical approaches for studying the yellow stripe-like transporter family in plants.

Iron (Fe) is essential for plants but can be toxic if over-accumulated. Members of the yellow stripe-like (YSL) family of metal transporters play important roles in plant Fe homeostasis, and a great deal of evidence has been gathered over many years that indicates the importance of YSLs in the long distance transport of metals complexed with nicotianamine (NA). This review examines our current knowledge of YSLs, gleaned from both genetic and biochemical approaches. Many unanswered questions remain regarding the substrate specificities of these transporters, which seem to vary widely depending on the individual transporter. Data are also just beginning to become available regarding YSLs in the most basal clade, which may be responsible for intracellular transport of metal-NA complexes. Future research on YSL transporters should focus on utilizing the proven techniques of yeast complementation and Xenopus oocyte electrophysiology to examine the substrate specificity of YSLs in greater detail.

CAN OF SPINACH, a novel long non-coding RNA, affects iron deficiency responses in Arabidopsis thaliana.

Long non-coding RNAs (lncRNAs) are RNA molecules with functions independent of any protein-coding potential. A whole transcriptome (RNA-seq) study of Arabidopsis shoots under iron sufficient and deficient conditions was carried out to determine the genes that are iron-regulated in the shoots. We identified two previously unannotated transcripts on chromosome 1 that are significantly iron-regulated. We have called this iron-regulated lncRNA, CAN OF SPINACH (COS). cos mutants have altered iron levels in leaves and seeds. Despite the low iron levels in the leaves, cos mutants have higher chlorophyll levels than WT plants. Moreover, cos mutants have abnormal development during iron deficiency. Roots of cos mutants are longer than those of WT plants, when grown on iron deficient medium. In addition, cos mutant plants accumulate singlet oxygen during iron deficiency. The mechanism through which COS affects iron deficiency responses is unclear, but small regions of sequence similarity to several genes involved in iron deficiency responses occur in COS, and small RNAs from these regions have been detected. We hypothesize that COS is required for normal adaptation to iron deficiency conditions.

Successful reproduction requires the function of Arabidopsis Yellow Stripe-Like1 and Yellow Stripe-Like3 metal-nicotianamine transporters in both vegetative and reproductive structures.

Several members of the Yellow Stripe-Like (YSL) family of proteins are transporters of metals that are bound to the metal chelator nicotianamine or the related set of mugineic acid family chelators known as phytosiderophores. Here, we examine the physiological functions of three closely related Arabidopsis (Arabidopsis thaliana) YSL family members, AtYSL1, AtYSL2, and AtYSL3, to elucidate their role(s) in the allocation of metals into various organs of Arabidopsis. We show that AtYSL3 and AtYSL1 are localized to the plasma membrane and function as iron transporters in yeast functional complementation assays. By using inflorescence grafting, we show that AtYSL1 and AtYSL3 have dual roles in reproduction: their activity in the leaves is required for normal fertility and normal seed development, while activity in the inflorescences themselves is required for proper loading of metals into the seeds. We further demonstrate that the AtYSL1 and AtYSL2 proteins, when expressed from the AtYSL3 promoter, can only partially rescue the phenotypes of a ysl1ysl3 double mutant, suggesting that although these three YSL transporters are closely related and have similar patterns of expression, they have distinct activities in planta. In particular, neither AtYSL1 nor AtYSL2 is able to functionally complement the reproductive defects exhibited by ysl1ysl3 double mutant plants.

Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters.

BACKGROUND AND AIMS: Brachypodium distachyon is a temperate grass with a small stature, rapid life cycle and completely sequenced genome that has great promise as a model system to study grass-specific traits for crop improvement. Under iron (Fe)-deficient conditions, grasses synthesize and secrete Fe(III)-chelating agents called phytosiderophores (PS). In Zea mays, Yellow Stripe1 (ZmYS1) is the transporter responsible for the uptake of Fe(III)-PS complexes from the soil. Some members of the family of related proteins called Yellow Stripe-Like (YSL) have roles in internal Fe translocation of plants, while the function of other members remains uninvestigated. The aim of this study is to establish brachypodium as a model system to study Fe homeostasis in grasses, identify YSL proteins in brachypodium and maize, and analyse their expression profiles in brachypodium in response to Fe deficiency. METHODS: The YSL family of proteins in brachypodium and maize were identified based on sequence similarity to ZmYS1. Expression patterns of the brachypodium YSL genes (BdYSL genes) were determined by quantitative RT-PCR under Fe-deficient and Fe-sufficient conditions. The types of PS secreted, and secretion pattern of PS in brachypodium were analysed by high-performance liquid chromatography. KEY RESULTS: Eighteen YSL family members in maize and 19 members in brachypodium were identified. Phylogenetic analysis revealed that some YSLs group into a grass-specific clade. The Fe status of the plant can regulate expression of brachypodium YSL genes in both shoots and roots. 3-Hydroxy-2'-deoxymugineic acid (HDMA) is the dominant type of PS secreted by brachypodium, and its secretion is diurnally regulated. CONCLUSIONS: PS secretion by brachypodium parallels that of related crop species such as barley and wheat. A single grass species-specific YSL clade is present, and expression of the BdYSL members of this clade could not be detected in shoots or roots, suggesting grass-specific functions in reproductive tissues. Finally, the Fe-responsive expression profiles of several YSLs suggest roles in Fe homeostasis.

WITHDRAWN: TcJAMYC: A bHLH transcription factor that activates paclitaxel biosynthetic pathway genes in yew.

This article was withdrawn by the authors before final publication on October 1, 2009.

Time to pump iron: iron-deficiency-signaling mechanisms of higher plants.

Iron is an essential nutrient for plants, yet it often limits plant growth. On the contrary, overaccumulation of iron within plant cells leads to oxidative stress. As a consequence, iron-uptake systems are carefully regulated to ensure that iron homeostasis is maintained. In response to iron limitation, plants induce expression of sets of activities that function at the root-soil interface to solubilize iron and subsequently transfer it across the plasma membrane of root cells. Recent advances have revealed key players in the signaling pathways that function to induce these iron-uptake responses. Transcription factors belonging to the basic helix-loop-helix, ABI3/VP1(B3), and NAC families appear to function either directly or indirectly in the upregulation of iron deficiency responses.

Analysis of Yellow Striped Mutants of Zea mays Reveals Novel Loci Contributing to Iron Deficiency Chlorosis.

The micronutrient iron (Fe) is essential for photosynthesis, respiration, and many other processes, but it is only sparingly soluble in aqueous solution, making adequate acquisition by plants a serious challenge. Fe is a limiting factor for plant growth on approximately 30% of the world's arable lands. Moreover, Fe deficiency in humans is a global health issue, affecting 1.62 billion people, or about 25% of the world's population. It is imperative that we gain a better understanding of the mechanisms that plants use to regulate iron homeostasis, since these will be important targets for future biofortification and crop improvement strategies. Grasses and non-grasses have evolved independent mechanisms for primary iron uptake from the soil. The grasses, which include most of the world's staple grains, have evolved a distinct 'chelation' mechanism to acquire iron from the soil. Strong iron chelators called phytosiderophores (PSs) are synthesized by grasses and secreted into the rhizosphere where they bind and solubilize Fe(III). The Fe(III)-PS complex is then taken up into root cells via transporters specific for the Fe(III)-PS complex. In this study, 31 novel, uncharacterized striped maize mutants available through the Maize Genetics Cooperation Stock Center (MGCSC) were analyzed to determine whether their mutant phenotypes are caused by decreased iron. Many of these proved to be either pale yellow or white striped mutants. Complementation tests were performed by crossing the MGCSC mutants to ys1 and ys3 reference mutants. This allowed assignment of 10 ys1 alleles and 4 ys3 alleles among the novel mutants. In addition, four ys∗ mutant lines were identified that are not allelic to either ys1 or ys3. Three of these were characterized as being non-allelic to each other and as having low iron in leaves. These represent new genes involved in iron acquisition by maize, and future cloning of these genes may reveal novel aspects of the grass iron acquisition mechanism.

Development of a particle bombardment-mediated transient transformation system for Taxus spp. cells in culture.

In developing and developed nations, plant cell culture systems are used to supply desirable compounds in lieu of chemical synthesis or natural extraction. When plant cell culture systems are unable to meet commercial demand, metabolic engineering offers a method to increase yields. However, to benefit from metabolic engineering approaches, effective transient transformation methods are required to rapidly identify and characterize key regulatory genes before intensive, time-consuming stable transformation efforts can proceed. This paper describes a particle bombardment-mediated transient transformation system for Taxus spp. in cell culture. Optimal parameters were established for the T. cuspidata cell line P991 and the T. canadensis cell line CO93D, resulting in reliable, efficient, transient expression of the firefly luciferase gene under control of the constitutive CaMV 35S promoter. Multiple bombardments and larger gold microcarriers (1.6 vs 1.0 microm in diameter) were particularly effective in increasing luciferase activity and in reducing variation among replicates. This particle bombardment-mediated transformation system was also shown to be capable of transiently expressing the DsRed and beta-glucuronidase reporter genes under the control of the maize ubiquitin and CaMV 35S promoters, respectively. With the ability to transiently transform Taxus spp. cell cultures using a variety of promoters and reporters, characterization of genes related to paclitaxel accumulation in culture can now proceed.

Jasmonate-responsive expression of paclitaxel biosynthesis genes in Taxus cuspidata cultured cells is negatively regulated by the bHLH transcription factors TcJAMYC1, TcJAMYC2, and TcJAMYC4.

Taxus cell suspension culture is a sustainable technology for the industrial production of paclitaxel (Taxol®), a highly modified diterpene anti-cancer agent. The methyl jasmonate (MJ)-mediated paclitaxel biosynthetic pathway is not fully characterized, making metabolic engineering efforts difficult. Here, promoters of seven genes (TASY, T5αH, DBAT, DBBT, PAM, BAPT, and DBTNBT), encoding enzymes of the paclitaxel biosynthetic pathway were isolated and used to drive MJ-inducible expression of a GUS reporter construct in transiently transformed Taxus cells, showing that elicitation of paclitaxel production by MJ is regulated at least in part at the level of transcription. The paclitaxel biosynthetic pathway promoters contained a large number of E-box sites (CANNTG), similar to the binding sites for the key MJ-inducible transcription factor AtMYC2 from Arabidopsis thaliana. Three MJ-inducible MYC transcription factors similar to AtMYC2 (TcJAMYC1, TcJAMYC2, and TcJAMYC4) were identified in Taxus. Transcriptional regulation of paclitaxel biosynthetic pathway promoters by transient over expression of TcJAMYC transcription factors indicated a negative rather than positive regulatory role of TcJAMYCs on paclitaxel biosynthetic gene expression.

Contribution of taxane biosynthetic pathway gene expression to observed variability in paclitaxel accumulation in Taxus suspension cultures.

Variability in product accumulation is one of the major obstacles limiting the widespread commercialization of plant cell culture technology to supply natural product pharmaceuticals. Despite extensive process engineering efforts, which have led to increased yields, plant cells exhibit variability in productivity that is poorly understood. Elicitation of Taxus cultures with methyl jasmonate (MeJA) induces paclitaxel accumulation, but to varying extents in different cultures. In the current study, cultures with different aggregation profiles were established to create predictable differences in paclitaxel accumulation upon MeJA elicitation. Expression of known paclitaxel biosynthetic genes in MeJA-elicited cultures exhibiting both substantial (15-fold) and moderate (2-fold) differences in paclitaxel accumulation was analyzed using quantitative reverse transcriptase PCR. Each population exhibited the characteristic large increase in paclitaxel pathway gene expression following MeJA elicitation; however, differences in expression between populations were minor, and only observed for the cultures with the 15-fold variation in paclitaxel content. These data suggest that although upregulation of biosynthetic pathway gene expression contributes to observed increases in paclitaxel synthesis upon elicitation with MeJA, there are additional factors that need to be uncovered before paclitaxel productivity can be fully optimized.

The role of transition metal homeostasis in plant seed development.

For human health, transition metal accumulation in edible seeds like cereal grains is of worldwide importance, since Fe and Zn deficiencies are among the most prevalent human nutritional disorders in the world. There have been many recent developments in our understanding of the patterns in which transition metals accumulate in the seeds, the identity of some specific transporters that are required for efficient seed metal accumulation, and the central role played by the ubiquitous plant metal chelator nicotianamine (NA). These and other recent discoveries will be reviewed here.

Transporters contributing to iron trafficking in plants.

This review will discuss recent progress in understanding the many roles of transporters in the whole-plant physiological processes that maintain iron (Fe) homeostasis. These processes include uptake from the soil via roots, control of transport from roots to above-ground parts of the plant, unloading of Fe from the xylem in above-ground parts, loading of Fe into mitochondria and plastids, transport of Fe to reproductive parts of the plant, and Fe mobilization during seed germination. In addition, we will discuss the mechanisms that plants use to cope with an apparently unintended consequence of Fe acquisition: the uptake of toxic heavy metals via Fe transporters. Rapid progress has been made in understanding the transport processes involved in each of these areas in the last 5 years and this review will focus on this recent progress. We will also highlight the key questions regarding transport steps that remain to be elucidated.

Disruption of OsYSL15 leads to iron inefficiency in rice plants.

Uptake and translocation of metal nutrients are essential processes for plant growth. Graminaceous species release phytosiderophores that bind to Fe(3+); these complexes are then transported across the plasma membrane. We have characterized OsYSL15, one of the rice (Oryza sativa) YS1-like (YSL) genes that are strongly induced by iron (Fe) deficiency. The OsYSL15 promoter fusion to beta-glucuronidase showed that it was expressed in all root tissues when Fe was limited. In low-Fe leaves, the promoter became active in all tissues except epidermal cells. This activity was also detected in flowers and seeds. The OsYSL15:green fluorescent protein fusion was localized to the plasma membrane. OsYSL15 functionally complemented yeast strains defective in Fe uptake on media containing Fe(3+)-deoxymugineic acid and Fe(2+)-nicotianamine. Two insertional osysl15 mutants exhibited chlorotic phenotypes under Fe deficiency and had reduced Fe concentrations in their shoots, roots, and seeds. Nitric oxide treatment reversed this chlorosis under Fe-limiting conditions. Overexpression of OsYSL15 increased the Fe concentration in leaves and seeds from transgenic plants. Altogether, these results demonstrate roles for OsYSL15 in Fe uptake and distribution in rice plants.

Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering.

Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol) for the treatment of many cancers. To develop an optimal bioprocess for paclitaxel supply, taxane biosynthetic pathway regulation must be better understood. Here we examine the expression profile of paclitaxel biosynthetic pathway genes by RNA gel blot analysis and RT-PCR in the Taxus cuspidata cell line P991 and compare with taxane metabolite levels. Upon methyl jasmonate (MJ) elicitation (100 microM), paclitaxel accumulates to 3.3 mg/L and cephalomannine to 2.2 mg/L 7 days after elicitation but neither are observed before this time. 10-deacetylbaccatin III accumulates to 3.3 mg/L and baccatin III to 1.2 mg/L by day 7 after elicitation. The early pathway enzyme genes GGPPS, TASY, and T5alphaH are up-regulated by MJ elicitation within 6 h and continue through 24 h before their abundances decrease. This study reveals the preference for one side of the biosynthetic pathway branch in early taxane synthesis, where transcripts coding for TalphaH are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch (TDAT and T10betaH) are not highly expressed following elicitation. Transcripts encoding the enzymes DBBT and DBAT are up-regulated upon MJ elicitation. Their products, 10-deacetylbaccatin III and baccatin III, respectively, accumulate within 6 h of the initial increase in transcript abundance. Importantly, the steady-state levels of the two terminal enzyme transcripts (BAPT and DBTNBT) are much lower than transcripts of early pathway steps. These are potential steps in the pathway for targeted metabolic engineering to increase accumulation of paclitaxel in suspension cell culture.

Mutations in Arabidopsis yellow stripe-like1 and yellow stripe-like3 reveal their roles in metal ion homeostasis and loading of metal ions in seeds.

Here, we describe two members of the Arabidopsis (Arabidopsis thaliana) Yellow Stripe-Like (YSL) family, AtYSL1 and AtYSL3. The YSL1 and YSL3 proteins are members of the oligopeptide transporter family and are predicted to be integral membrane proteins. YSL1 and YSL3 are similar to the maize (Zea mays) YS1 phytosiderophore transporter (ZmYS1) and the AtYSL2 iron (Fe)-nicotianamine transporter, and are predicted to transport metal-nicotianamine complexes into cells. YSL1 and YSL3 mRNAs are expressed in both root and shoot tissues, and both are regulated in response to the Fe status of the plant. Beta-glucuronidase reporter expression, driven by YSL1 and YSL3 promoters, reveals expression patterns of the genes in roots, leaves, and flowers. Expression was highest in senescing rosette leaves and cauline leaves. Whereas the single mutants ysl1 and ysl3 had no visible phenotypes, the ysl1ysl3 double mutant exhibited Fe deficiency symptoms, such as interveinal chlorosis. Leaf Fe concentrations are decreased in the double mutant, whereas manganese, zinc, and especially copper concentrations are elevated. In seeds of double-mutant plants, the concentrations of Fe, zinc, and copper are low. Mobilization of metals from leaves during senescence is impaired in the double mutant. In addition, the double mutant has reduced fertility due to defective anther and embryo development. The proposed physiological roles for YSL1 and YSL3 are in delivery of metal micronutrients to and from vascular tissues.