Genome-wide and molecular evolution analysis of the Poplar KT/HAK/KUP potassium transporter gene family

As the largest K(+) transport gene family, KT/HAK/KUP family plays an important role in plant growth, development, and stress adaptation. However, there is limited information about this family in woody plant species. In this study, with genome-wide in-depth investigation, 31 Poplar KT/HAK/KUP transporter genes including six pairs of tandem duplicated and eight pairs of segmental duplicated paralogs have been identified, suggesting segmental and tandem duplication events contributed to the expansion of this family in Poplar. The combination of phylogenetic, exon structure and splice site, and paragon analysis revealed 11 pairs of Poplar KT/HAK/KUP duplicates. For these 11 pairs, all pairs are subject to purify selection, and asymmetric evolutionary rates have been found to occur in three pairs. This study might provide more insights into the underlying evolution mechanisms of trees acclimating to their natural habitat.     

钾离子转运载体HAK/KUP/KT家族参与植物耐盐性的研究进展

钾可以通过多种方式参与植物的生长和发育,在植物缓解盐等非生物胁迫方面发挥重要作用。在植物中,HAK/KUP/KT是成员数目最多的一类高亲和钾转运蛋白家族,本文对该家族成员的分类、盐胁迫下钾的吸收、转运、生理功能和分子调控机制等方面的研究进行了综述,并对该转运体家族今后的研究方向进行了展望。     

Plant KT/KUP/HAK potassium transporters: single family - multiple functions

BACKGROUND AND AIMS: Potassium transporters belonging to the KT/KUP/HAK family are important for various aspects of plant life including mineral nutrition and the regulation of development. Genes encoding these transporters are present in the genomes of all plants, but have not been found in the genomes of Protista or Animalia. The aim of this Botanical Briefing is to analyse the function of KT/KUP/HAK transporters from evolutionary, molecular and physiological perspectives. SCOPE: This Briefing covers the phylogeny and evolution of KT/KUP/HAK transporters, the role of transporters in plant mineral nutrition and potassium homeostasis, and the role of KT/KUP/HAK transporters in plant development.     

Functional HAK/KUP/KT-like potassium transporter encoded by chlorella viruses

Chlorella viruses are a source of interesting membrane transport proteins. Here we examine a putative K(+) transporter encoded by virus FR483 and related chlorella viruses. The protein shares sequence and structural features with HAK/KUP/KT-like K(+) transporters from plants, bacteria and fungi. Yeast complementation assays and Rb(+) uptake experiments show that the viral protein, termed HAKCV (high-affinity K(+) transporter of chlorella virus), is functional, with transport characteristics that are similar to those of known K(+) transporters. Expression studies revealed that the protein is expressed as an early gene during viral replication, and proteomics data indicate that it is not packaged in the virion. The function of HAKCV is unclear, but the data refute the hypothesis that the transporter acts as a substitute for viral-encoded K(+) channels during virus infection.     

Knockout of the AtCESA2 gene affects microtubule orientation and causes abnormal cell expansion in Arabidopsis

Complete cellulose synthesis is required to form functional cell walls and to facilitate proper cell expansion during plant growth. AtCESA2 is a member of the cellulose synthase A family in Arabidopsis (Arabidopsis thaliana) that participates in cell wall formation. By analysis of transgenic seedlings, we demonstrated that AtCESA2 was expressed in all organs, except root hairs. The atcesa2 mutant was devoid of AtCESA2 expression, leading to the stunted growth of hypocotyls in seedlings and greatly reduced seed production in mature plants. These observations were attributed to alterations in cell size as a result of reduced cellulose synthesis in the mutant. The orientation of microtubules was also altered in the atcesa2 mutant, which was clearly observed in hypocotyls and petioles. Complementary expression of AtCESA2 in atcesa2 could rescue the mutant phenotypes. Together, we conclude that disruption of cellulose synthesis results in altered orientation of microtubules and eventually leads to abnormal plant growth. We also demonstrated that the zinc finger-like domain of AtCESA2 could homodimerize, possibly contributing to rosette assemblies of cellulose synthase A within plasma membranes.     

Molecular cloning and expression analysis of a gene encoding KUP/HAK/KT-type potassium uptake transporter from Cryptomeria japonica

Key message

The molecular mechanism of potassium ion transport across membranes in conifers is poorly known. We isolated and analyzed a gene encoding a potassium transporter from the conifer Cryptomeria japonica.

Abstract

Potassium ion (K⁺) is an essential and the most abundant intracellular cation in plants. The roles of K⁺ in various aspects of plant life are closely linked to its transport across biological membranes such as the plasma membrane and the tonoplast, which is mediated by membrane-bound transport proteins known as transporters and channels. Information on the molecular basis of K⁺ membrane transport in trees, especially in conifers, is currently limited. In this study, we isolated one complementary DNA, CjKUP1, which is homologous to known plant K⁺ transporters, from Cryptomeria japonica. Complementation tests using an Escherichia coli mutant, which is deficient in K⁺ uptake activity, was conducted to examine the K⁺ uptake function of the protein encoded by CjKUP1. Transformation of the K⁺-uptake-deficient mutant with CjKUP1 complemented the deficiency of this mutant. This result indicates that CjKUP1 has a function of K⁺ uptake. The expression levels of CjKUP1 in male strobili were markedly higher from late September to early October than in other periods. The expression levels in male and female strobili were higher than those in other organs such as needles, inner bark, differentiating xylem, and roots. These results indicate that CjKUP1 is mainly involved in K⁺ membrane transport in the cells of reproductive organs of C. japonica trees, especially in male strobili during pollen differentiation.     

Tryptophan deficiency affects organ growth by retarding cell expansion in Arabidopsis

Tryptophan (Trp) is an essential amino acid required not only for protein synthesis but also for the production of many plant metabolites, including the hormone auxin. Mutations that disrupt Trp biosynthesis result in various developmental defects in plant organs, but how Trp affects organ growth and development remains unclear. Here, we identify an Arabidopsis mutant, small organ1 ( smo1/trp2-301 ), which exhibits a reduction in the size of its aerial organs as a result of the retardation of growth by cell expansion, rather than by the retardation of growth by cell proliferation. smo1/trp2-301 contains a lesion in TSB1 that encodes a predominantly expressed Trp synthase β-subunit, and is allelic with trp2 mutants. Further analyses show that in trp2 leaf cells, the nuclear endoreduplication is impaired and chloroplast development is delayed. Furthermore, cell expansion and leaf growth in trp2 can be restored by the exogenous application of Trp, but not by auxin, and the general protein synthesis is not apparently affected in trp2 mutants. Our findings suggest that the deficiency in Trp or its derivatives is a growth-limiting factor for cell expansion during plant organogenesis.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem

Postembryonic organ formation in higher plants relies on the activity of stem cell niches in shoot and root meristems where differentiation of the resident cells is repressed by signals from surrounding cells. We searched for mutations affecting stem cell maintenance and isolated the semidominant l28 mutant, which displays premature termination of the shoot meristem and differentiation of the stem cells. Allele competition experiments suggest that l28 is a dominant-negative allele of the APETALA2 (AP2) gene, which previously has been implicated in floral patterning and seed development. Expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) genes, which regulate stem cell maintenance in the wild type, were disrupted in l28 shoot apices from early stages on. Unlike in floral patterning, AP2 mRNA is active in the center of the shoot meristem and acts via a mechanism independent of AGAMOUS, which is a repressor of WUS and stem cell maintenance in the floral meristem. Genetic analysis shows that termination of the primary shoot meristem in l28 mutants requires an active CLV signaling pathway, indicating that AP2 functions in stem cell maintenance by modifying the WUS-CLV3 feedback loop.     

Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis

Members of the AKT2/3 family have been identified as photosynthate-induced phloem K(+) channels. Here we describe the isolation and characterisation of an AKT2/3 loss-of-function mutant (akt2/3-1) from Arabidopsis thaliana (L.) Heynh. Microautoradiography following (14)CO(2) incubation in the light revealed that a major fraction of (14)CO(2)-derived photosynthates leaking out of sieve tubes appears not to be effectively reloaded (retrieval) into the phloem of the mutant. Using the aphid stylectomy technique we showed that the phloem sap of the mutant, lacking the phloem channels of the AKT2/3 type, contained only half the sucrose content of the wild type. Furthermore, the akt2/3-1 mutant exhibited a reduced K(+) dependence of the phloem potential. Xenopus oocytes expressing the phloem sucrose/proton symporter depolarise upon sucrose application. When, however, the phloem channel was co-expressed - mimicking the situation in the sieve tube/companion cell complex - depolarisation was prevented. From our studies we thus conclude that AKT2/3 regulates the sucrose/H(+) symporters via the phloem potential.     

A hypermorphic mutation in the protein phosphatase 2C HAB1 strongly affects ABA signaling in Arabidopsis

Protein phosphatases of the 2C family (PP2C) function in the regulation of several signaling pathways from prokaryotes to eukaryotes. In Arabidopsis thaliana, the HAB1 PP2C is a negative regulator of the stress hormone abscisic acid (ABA) signaling. Here, we show that plants expressing a mutant form of HAB1 in which Gly246 was mutated to Asp (G246D) display strong ABA insensitive phenotypes. Our results indicate that the G246D mutation has a hypermorphic rather than a dominant negative effect. The data suggest that this mutation localized in a conserved motif in the PP2C catalytic domain could be used in other PP2Cs to reveal their biological functions.     

Regulation of the cell expansion gene RHD3 during Arabidopsis development

The RHD3 (ROOT HAIR DEFECTIVE3) gene encodes a putative GTP-binding protein required for appropriate cell enlargement in Arabidopsis. To obtain insight into the mechanisms of RHD3 regulation, we conducted a molecular genetic dissection of RHD3 gene expression and function. Gene fusion and complementation studies show that the RHD3 gene is highly expressed throughout Arabidopsis development and is controlled by two major regulatory regions. One regulatory region is located between -1,500 and -600 bp upstream of the RHD3 gene and is required for vascular tissue expression. The other region is intragenically located and includes the 558-bp first intron, which is responsible for high-level expression of RHD3 throughout the plant. The presence and location of this intron is essential for gene function because constructs lacking this intron or constructs with the intron in an abnormal position are unable to functionally complement the rhd3 mutations. We also analyzed the role of other RHD genes and the plant hormones auxin and ethylene in RHD3 regulation, and we determined that these act downstream or independently from the RHD3 pathway. This study shows that multiple levels of regulation are employed to ensure the appropriate expression of RHD3 throughout Arabidopsis development.     

Arabidopsis IRT2 gene encodes a root-periphery iron transporter

Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.     

Genome-wide and molecular evolution analyses of the KT/HAK/KUP family in tomato (Solanum lycopersicum L.)

Potassium transporters belonging to the KT/HAK/KUP family play an important role in plant growth, development, mineral nutrition, and stress adaptation. In this study, we identified 19 KT/HAK/KUP family genes in tomato, distributed on 10 chromosomes, by using bioinformatics methods. A complete overview of the KT/HAK/KUP (SlHAK) genes in tomato is presented, including chromosome location, phylogeny, gene structure, and evolution pattern. Phylogenetic analysis of 19 SlHAK proteins suggested that group IV of the KT/HAK/KUP family is absent in the tomato genome. In addition, five pairs of segmental duplicated paralogs and two pairs of tandem duplicated paralogs were identified in the tomato KT/HAK/KUP family. This suggests that segmental duplication is predominant for the expansion of the SlHAK genes. Calculation of the approximate dates of duplication events using the synonymous substitution rate indicated that the segmental duplication of the KT/HAK/KUP genes in tomato originated 35.89–62.77 million years ago. Adaptive evolution analysis showed that purifying selection contributed to the evolution of segmental duplicated pairs. Furthermore, Tajima’s relative rate test indicated that all segmental duplicated pairs evolved at similar rates. As a first step toward a genome-wide analysis of the KT/HAK/KUP gene family in tomato, our results provide valuable information for understanding the function and evolution of the KT/HAK/KUP gene family in tomato and other species.