VLN2 Regulates Plant Architecture by Affecting Microfilament Dynamics and Polar Auxin Transport in Rice

As a fundamental and dynamic cytoskeleton network, microfilaments (MFs) are regulated by diverse actin binding proteins (ABPs). Villins are one type of ABPs belonging to the villin/gelsolin superfamily, and their function is poorly understood in monocotyledonous plants. Here, we report the isolation and characterization of a rice (Oryza sativa) mutant defective in VILLIN2 (VLN2), which exhibits malformed organs, including twisted roots and shoots at the seedling stage. Cellular examination revealed that the twisted phenotype of the vln2 mutant is mainly caused by asymmetrical expansion of cells on the opposite sides of an organ. VLN2 is preferentially expressed in growing tissues, consistent with a role in regulating cell expansion in developing organs. Biochemically, VLN2 exhibits conserved actin filament bundling, severing and capping activities in vitro, with bundling and stabilizing activity being confirmed in vivo. In line with these findings, the vln2 mutant plants exhibit a more dynamic actin cytoskeleton network than the wild type. We show that vln2 mutant plants exhibit a hypersensitive gravitropic response, faster recycling of PIN2 (an auxin efflux carrier), and altered auxin distribution. Together, our results demonstrate that VLN2 plays an important role in regulating plant architecture by modulating MF dynamics, recycling of PIN2, and polar auxin transport

VLN2

As a fundamental and dynamic cytoskeleton network, microfilaments (MFs) are regulated by diverse actin binding proteins (ABPs). Villins are one type of ABPs belonging to the villin/gelsolin superfamily, and their function is poorly understood in monocotyledonous plants. Here, we report the isolation and characterization of a rice (Oryza sativa) mutant defective in VILLIN2 (VLN2), which exhibits malformed organs, including twisted roots and shoots at the seedling stage. Cellular examination revealed that the twisted phenotype of the vln2 mutant is mainly caused by asymmetrical expansion of cells on the opposite sides of an organ. VLN2 is preferentially expressed in growing tissues, consistent with a role in regulating cell expansion in developing organs. Biochemically, VLN2 exhibits conserved actin filament bundling, severing and capping activities in vitro, with bundling and stabilizing activity being confirmed in vivo. In line with these findings, the vln2 mutant plants exhibit a more dynamic actin cytoskeleton network than the wild type. We show that vln2 mutant plants exhibit a hypersensitive gravitropic response, faster recycling of PIN2 (an auxin efflux carrier), and altered auxin distribution. Together, our results demonstrate that VLN2 plays an important role in regulating plant architecture by modulating MF dynamics, recycling of PIN2, and polar auxin transport

<em>Ehd4</em> Encodes a Novel and <em>Oryza</em>-Genus-Specific Regulator of Photoperiodic Flowering in Rice

<div><p>Land plants have evolved increasingly complex regulatory modes of their flowering time (or heading date in crops). Rice (<i>Oryza sativa</i> L.) is a short-day plant that flowers more rapidly in short-day but delays under long-day conditions. Previous studies have shown that the <i>CO</i>-<i>FT</i> module initially identified in long-day plants (Arabidopsis) is evolutionary conserved in short-day plants (<i>Hd1</i>-<i>Hd3a</i> in rice). However, in rice, there is a unique <i>Ehd1</i>-dependent flowering pathway that is <i>Hd1</i>-independent. Here, we report isolation and characterization of a positive regulator of <i>Ehd1</i>, <i>Early heading date 4</i> (<i>Ehd4</i>). <i>ehd4</i> mutants showed a never flowering phenotype under natural long-day conditions. Map-based cloning revealed that <i>Ehd4</i> encodes a novel CCCH-type zinc finger protein, which is localized to the nucleus and is able to bind to nucleic acids <i>in vitro</i> and transactivate transcription in yeast, suggesting that it likely functions as a transcriptional regulator. <i>Ehd4</i> expression is most active in young leaves with a diurnal expression pattern similar to that of <i>Ehd1</i> under both short-day and long-day conditions. We show that <i>Ehd4</i> up-regulates the expression of the “florigen” genes <i>Hd3a</i> and <i>RFT1</i> through <i>Ehd1,</i> but it acts independently of other known <i>Ehd1</i> regulators. Strikingly, <i>Ehd4</i> is highly conserved in the <i>Oryza</i> genus including wild and cultivated rice, but has no homologs in other species, suggesting that <i>Ehd4</i> is originated along with the diversification of the <i>Oryza</i> genus from the grass family during evolution. We conclude that <i>Ehd4</i> is a novel <i>Oryza</i>-genus-specific regulator of <i>Ehd1</i>, and it plays an essential role in photoperiodic control of flowering time in rice.</p> </div

Expression pattern of <i>Ehd4</i>.

<p>(<i>A</i>) 30-d old wild-type plants (Kita-ake) grown under SDs were used for quantitative RT-PCR. DL1, newly emerging leaf; DL2, expending leaf; DL3, fully expended leaf; ASA, around the shoot apex. (<i>B</i>) <i>Ehd4</i> transcript levels in various organs (means±s.d, <i>n</i> = 3). (<i>C</i>) to (<i>I</i>) GUS staining of various organs in <i>pEHD4::GUS</i> transgenic plants. (<i>C</i>) Root; (<i>D</i>) Floret; (<i>E</i>) Stem; (<i>F</i>) to (<i>H</i>) Transverse sections of stem, immature leaf and sheath, respectively; (<i>I</i>) Longitudinal section of the shoot apical meristem (SAM). Arrow indicates phloem in (<i>F</i>) and (<i>G</i>) and SAM in (<i>I</i>). (<i>J</i>) and (<i>K</i>) Rhythmic and developmental expression of <i>Ehd4</i>. The rice <i>Ubiquitin-1</i> (<i>UBQ</i>) gene was used as the internal control. Values are shown as mean±s.d of three independent experiments and two biological replicates. The open and filled bars at the bottom represent the light and dark periods, respectively. s.d: standard deviations.</p

Map-based cloning of <i>Ehd4</i>.

<p>(<i>A</i>) Location of the <i>Ehd4</i> locus on rice chromosome 3. (<i>B</i>) High-resolution linkage map of <i>Ehd4</i>. (<i>C</i>) Candidate genes on BAC OSJNBb0005F16. (<i>D</i>) Structure of the <i>Ehd4</i> gene. Lines, black and white boxes represent introns, exons and untranslated regions, respectively. The base change from G to A creates an early stop codon (Asterisk). (<i>E</i>) Complementation of <i>ehd4</i>. <i>Ehd4</i> was driven by either the native promoter (<i>pEhd4::Ehd4</i>) or the maize <i>Ubiquitin-1</i> promoter (<i>pUbi::Ehd4</i>). T2 plants of two <i>pEhd4::Ehd4</i> lines (#26 and #34) and two <i>pUbi::Ehd4</i> lines (#18 and #24) were measured (<i>n</i> = 10). All plants were grown under both SD and LD conditions.</p

Quantitative RT–PCR analysis of representative flowering-related genes and <i>Ehd4</i> in various flowering-time mutants or their NILs (near-isogenic lines) and corresponding WTs under SDs and LDs.

<p>(<i>A</i>) Transcript level of <i>Ehd2</i>, <i>Ehd3</i>, <i>OsMADS50</i>, <i>OsGI</i>, <i>OsMADS51</i>, <i>OsphyB</i>, <i>OsCOL4</i> and <i>DTH8</i> in WT (Kita-ake) and <i>ehd4</i> plants. (<i>B</i>) Transcript level of <i>Ghd7</i>, <i>Hd3a</i>, <i>RFT1</i>, <i>Ehd1</i> and <i>Hd1</i> in WT (Nipponbare) and <i>ehd4</i>-<i>Nip</i> plants. (<i>C</i>) Transcript level of <i>Ehd4</i> in various flowering-time mutants or their NILs (near-isogenic lines) and corresponding WTs. Dongjin and the <i>osphyb</i> mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Lee1" target="_blank">[26]</a>; Tohoku IL9 and the <i>ehd2</i> mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Matsubara1" target="_blank">[34]</a>; Tohoku IL9 and the <i>ehd3</i> mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Matsubara2" target="_blank">[36]</a>; Dongjin and the <i>osmads50</i> mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Lee2" target="_blank">[31]</a>; Dongjin and the <i>osmads51</i> mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Kim1" target="_blank">[30]</a>; Nipponbare and a NIL carrying a non-functional <i>Hd1</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Yano1" target="_blank">[19]</a>; <u>Aso</u>minori and a NIL carrying a nonfunctional <i>DTH8</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Wei1" target="_blank">[29]</a>; A NIL carrying a functional <i>Ghd7</i> allele and a NIL carrying a non-functional in the Shanyou 63 background <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Xue1" target="_blank">[28]</a>. Taichun 65 carrying a non-functional <i>Ehd1</i> allele and a NIL carrying a functional <i>Ehd1</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Doi1" target="_blank">[24]</a>; Nipponbare carrying a partially functional <i>Hd3a</i> allele and a NIL carrying a functional <i>Hd3a</i> allele <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003281#pgen.1003281-Kojima1" target="_blank">[20]</a>; Penultimate leaves were harvested around reported peak expression level of each gene during the 24 hrs photoperiod - at dawn for <i>OsphyB</i>, <i>OsCOL4</i>, <i>Ehd1</i>, <i>Ehd2</i>, <i>Hd3a</i>, <i>RFT1</i> and <i>Ehd4</i>, 3 h after dawn for <i>Ghd7</i>, 8 h after dawn for <i>Ehd3</i>, <i>OsMADS50</i>, <i>OsMADS51</i> and <i>DTH8</i> and immediately after dusk for <i>OsGI</i> and <i>Hd1</i> from 28 d-old (SDs) and 35 d-old (LDs) plants. The rice <i>Ubiquitin-1</i> (<i>UBQ</i>) gene was used as the internal control. Values are shown as mean±s.d (standard deviations) of three independent experiments and two biological replicates.</p

Natural variations in the <i>Ehd4</i> coding region among rice germplasm core collection.

<p>(<i>A</i>) Haplotype network of the <i>Ehd4</i> alleles in 86 rice accessions. Haplotype frequencies are proportional to the area of the circles. The proportion of wild rice and two cultivated subgroups (<i>indica</i> and <i>japonica</i>) in each haplotype is represented by different colors. (<i>B</i>) The polymorphic nucleotides of Hap_2 and Hap_3 of <i>Ehd4</i> gene in the core collection. The number on the top shows the position of nucleotide polymorphisms in the coding region starting from the ATG start codon. (<i>C</i>) Geographic distribution of the cultivated rice accessions belonging to Hap_2 and Hap_3. (<i>D</i>) Flowering time of transgenic plants carrying two major haplotypes of <i>Ehd4</i> driven by the maize <i>Ubiquitin-1</i> promoter in <i>ehd4</i> (Kita-ake background) and NIL carrying <i>Ehd4</i>^Hap3 compared with the 93-11 parental plants. T2 plants of two <i>pUbi::Ehd4</i>^Hap3 (#18 and #24) and two <i>pUbi::Ehd4</i>^Hap2 (#12 and #16) lines were measured (<i>n</i> = 15). All plants were grown in the natural long day field conditions. Values are means±s.d. (standard deviations) (<i>n</i> = 15). **Significant at 1% level; <i>n.s.</i>, not significant.</p