Isolation and Characterization of a Conserved Domain in the Eremophyte H⁺-PPase Family

<div><p>H⁺-translocating inorganic pyrophosphatases (H⁺-PPase) were recognized as the original energy donors in the development of plants. A large number of researchers have shown that H⁺-PPase could be an early-originated protein that participated in many important biochemical and physiological processes. In this study we cloned 14 novel sequences from 7 eremophytes: <i>Sophora alopecuroid</i> (<i>Sa</i>), <i>Glycyrrhiza uralensis</i> (<i>Gu</i>), <i>Glycyrrhiza inflata</i> (<i>Gi</i>), <i>Suaeda salsa</i> (<i>Ss</i>), S<i>uaeda rigida</i> (<i>Sr</i>), <i>Halostachys caspica</i> (<i>Hc</i>), and <i>Karelinia caspia</i> (<i>Kc</i>). These novel sequences included 6 ORFs and 8 fragments, and they were identified as H⁺-PPases based on the typical conserved domains. Besides the identified domains, sequence alignment showed that there still were two novel conserved motifs. A phylogenetic tree was constructed, including the 14 novel H⁺-PPase amino acid sequences and the other 34 identified H⁺-PPase protein sequences representing plants, algae, protozoans and bacteria. It was shown that these 48 H⁺-PPases were classified into two groups: type I and type II H⁺-PPase. The novel 14 eremophyte H⁺-PPases were classified into the type I H⁺-PPase. The 3D structures of these H⁺-PPase proteins were predicted, which suggested that all type I H⁺-PPases from higher plants and algae were homodimers, while other type I H⁺-PPases from bacteria and protozoans and all type II H⁺-PPases were monomers. The 3D structures of these novel H⁺-PPases were homodimers except for <i>SaVP3</i>, which was a monomer. This regular structure could provide important evidence for the evolutionary origin and study of the relationship between the structure and function among members of the H⁺-PPase family.</p></div

Tentative transmembrane model of H⁺-PPase from <i>Sophora alopecuroid</i> generated by TMHMM online.

<p>There are 13 putative transmembrane regions. D1, D2 and D3 with solid rectangles are previously identified domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070099#pone.0070099-Hedlund1" target="_blank">[26]</a>, and P1 and P2 with dotted rectangles are putative motifs that we predicted.</p

The sequence and transmembrane prediction of <i>SaVP1</i>.

<p>There are 13 transmembrane regions in <i>SaVP1</i> which are marked with red Arabic numerals. The yellow marked K is group I, and the green marked K is group II. The purple marked segment consists of identified conserved domains. The grey marked segment consists of supposed novel conserved motifs.</p

Phylogenetic tree of H⁺-PPase sequences from some representative species by NJ and a 3D structure prediction by Swiss Model.

<p>Purple indicates novel cloned H⁺-PPases from 7 eremophytes. The type I H⁺-PPases were made up of a, b, c and d subgroups. The a subgroup is made up of <i>ScVP</i> (ADQ00196.1), <i>HcVP</i> (ABO45933.1), <i>KfVP</i> (ABK91685.1), <i>ChrVP</i> (AAM97920.1), <i>SsVP2</i>, <i>HcVP3</i>, <i>MtVP</i> (XP_003609464.1), <i>SaVP1</i>, <i>SaVP2</i>, <i>GuVP1</i>, <i>GiVP1</i>, <i>GhVP</i> (ADN96173.1), <i>NtVP</i> (CAA54869.1), and <i>AVP1</i> (NP_173021.1). The b subgroup includes <i>OsVP</i> (BAD25066.1), <i>BvVP</i> (AAA61610.1), <i>HcVP</i>1, <i>HcVP</i>2, <i>SrVP</i>1, <i>SsVP</i>1, <i>GuVP</i>2, <i>KcVP1</i> and <i>KcVP2</i>. The c subgroup is formed by <i>BdVP</i> (XP_003564217.1), <i>ZmVP</i> (ACN33286.1), <i>SbVP</i> (ADJ67258.1), <i>ZxVP</i> (ABU92563.1), <i>GmVP</i> (XP_003555808.1), <i>PtVP</i> (XP_002318956.1), <i>RcVP</i> (XP_002512502.1). And the d is formed by <i>ChlrVP</i> (XP_001694682.1), SaVP3, <i>PbVP</i> (XP_676243.1), <i>RhmVP</i> (YP_004826142.1), <i>ChpVP</i> (YP_001959520.1), <i>ThmVP</i> (YP_003676510.1), <i>ElVP</i> (YP_003958643.1), <i>HhVP</i> (YP_003994656.1), <i>FvVP</i> (ZP_08694297.1). The type II H⁺-PPase includes <i>AVP2</i> (NM_101539), <i>MpVP</i> (XM_003059582.1), <i>RhrVP</i> (YP_426905.1), <i>MgVP</i> (CAM76045.1), <i>NeVP</i> (YP_747021.1), <i>GsVP</i> (NP_954331.1), <i>AbVP</i> (EKS38298.1), <i>RhpVP</i> (Q8KY01.1), and <i>MmVP</i> (YP_865668.1). 3D structures including type I H⁺-PPase <i>ScVP1</i> (ADQ00196.1, <i>Suaeda corniculata</i>), <i>OsVP1</i> (BAD25066.1, <i>Oryza sativa</i>), <i>ChrVP1</i> (XP_001694682.1, <i>Chlamydomonas reinhardtii</i>), <i>SrVP1</i>, <i>SaVP1</i> and <i>KcVP1</i> are present as homodimers. The others are monomer H⁺-PPase, they are <i>AVP2</i> (NM_101539, <i>Arabidopsis thaliana 2</i>), <i>SaVP3</i> and <i>MgVP2</i> (CAM76045.1, <i>Magnetospirillum gryphiswaldense</i>). The <i>SaVP1-1</i> was truncated according to the sequence of <i>SaVP3</i>. Number 1, 2 and 3 and black arrows in the 3D structure showed different regions.</p

Multiple sequence alignment with H⁺-PPase sequences isolated from 7 eremophytes.

<p>Two ORFs and a fragment were from <i>Sa</i> (<i>SaVP1</i>, <i>SaVP2</i> and <i>SaVP3</i>). The two ORFs were from <i>Gu</i> (<i>GuVP1</i> and <i>GuVP2</i>) and one ORF from <i>Gi</i> (<i>GiVP1</i>). The two fragments were from <i>Ss</i> (<i>SsVP1</i> and <i>SsVP2</i>), one fragment was from <i>Sr</i> (<i>SrVP1</i>), and three fragments were from <i>Hc</i> (<i>HcVP1</i>, <i>HcVP2</i> and <i>HcVP3</i>). The other ORF and fragment were from <i>Kc</i> (<i>KcVP1</i> and <i>KcVP2</i>). The sequences marked with solid underline are the identified conserved domains GGG, DVGADLVGK, DNVGDNVGD, TEYYT, and GNTTAA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070099#pone.0070099-Baltscheffsky2" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070099#pone.0070099-Drozdowicz2" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070099#pone.0070099-Hedlund1" target="_blank">[26]</a>. The putative motifs GPISDNAGGIAEM and FLLGGITSLISGFLGM were marked by dotted underline.</p

Consensus sequences of the conserved domains of H⁺-PPase that were isolated from 7 eremophytes and more than 240 H⁺-PPases from NCBI.

<p>The sequences IALFGRV<u>DGG</u>IYTKAA<u>DVGADLVGKVE</u>RNIPEDDPRNPAVIA<u>DNVG DNVGD</u>IAGMGSDL (A), T<u>EYYT</u>S (B), and A<u>GNTTAA</u>IGKGFAIGSAA (C) are previously identified conserved domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070099#pone.0070099-Baltscheffsky2" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070099#pone.0070099-Hedlund1" target="_blank">[26]</a>. The sequences FTAVAFLL<u>G</u>GIT<u>S </u>LIS<u>G</u>FL<u>GM</u>KI (D) and <u>GP</u>IS<u>DNAGGI</u>A<u>EM</u> (E) are new possible motifs, according to conserved sequence and structure.</p

Primers for RACE and ORF of the H⁺-PPases from the 7 eremophytes.

<p>Primers for RACE and ORF of the H⁺-PPases from the 7 eremophytes.</p

The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

<div><p>Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (<i>FLS)</i> genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (<i>ANR)</i> genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase <i>(F3H)</i> silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development.</p></div

The molecular phylogenetic analysis of the orthologous PME genes, and pro and PMEI domains.

<p>(A) The phylogenetic analysis of the pro and PMEI domains in 10 species using the neighbor joining method. The blue lines represent the pro domain, and the red lines represent the PMEI domain. (B) The consensus sequence alignment shows the conserved motifs of the pro and PMEI domains in the representative species. The MEME program was then used to verify the conserved motifs.</p

The Ka/Ks value distributions of the PME, pro and PMEI domains in 10 species.

<p>The red broken line indicates that genes are under positive selection (more than one) or negative selection (less than one). The short species names are <i>P</i><i>. patens</i> (Pp), <i>S</i><i>. moellendorffii</i> (Sm), <i>A</i><i>. trichopoda</i> (Am), <i>S.</i> bicolor (Sb), <i>O. sativa</i> (Os), <i>S</i><i>. lycopersicum</i> (Sl), <i>V. vinifera</i> (Vv), <i>P</i><i>. trichocarpa</i> (Pt), <i>C. papaya</i> (Cp) and <i>A. thaliana</i> (At). The Ka and Ks values were computed using the PAML program.</p