Auxins Regulations of Branched Spike Development and Expression of TFL , a LEAFY-Like Gene in Branched Spike Wheat ( Triticum aestivum )

Branched spike wheat is a hexaploid germplasm with branched rachis on its main rachises, and the crucial period for branched rachises occurrence and development is just after the two ridges stage of shoot apex. Natural [indole-3-acetic acid (IAA), indole-3butyric acid (IBA)] and synthetic [(1-naphthaleneacetic acid (NAA), 2,4-Dichlorophenoxyacetic acid (2,4-D)] auxins were applied at this period to investigate the spike traits, seedling growth and photosynthesis related characters and expression of a putative homologue of the LEAFY in branched spike wheat. The four types of experienced auxins induced similar effects on these foresaid characters, although the impact extents were different among the auxins treatments. More branched rachis, spikelets, fertile florets and longer branched rachis were obtained in plants with IAA and IBA at 0.1 mM or NAA and 2,4-D at 1.0mM than those plants with no auxin treated. Auxin treatments also increased fresh and dry mass, photosynthetic pigment and parameters. TFL, a LEAFY-like gene was cloned in branched spike wheat and TFL mRNA expression was quantified using real-time reverse transcriptase-PCR. Application of the auxins accelerated the rise in TFL expression during the periods of branched rachises occurrence and extension. The data supports the hypothesis that auxins play a central role in the regulation branched spike development and TFL might correlate with the development of branched rachises in branched spike wheat.


Introduction
Branched spike wheat is a special hexaploid wheat germplasm which has branches (branched rachises) on its main rachis (inflorescence axis) and bears an overabundance of spikelets and grains on a spike.The grain number in a branched spike can reach 70-130 while that in a single normal spike generally is 35-70.Branched spike wheat has great application potentials in wheat production areas with high speed of grain filling or long term of grain filling period.
A number of studies have been conducted on field performances of the branched spike wheat.The supernumerary spikelet character in this type of spike was affected by light, temperature, and nutrients Traditionally, plant hormones and synthetic plant growth regulators are used as valuable research tools to elucidated physiological responses of plant or probe biochemical control mechanisms.In many bioassays, it has been shown that auxins play an important role in plant growth and development (Cooke et al., 2002).Indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) are naturally-occurring, plant hormone of the auxin class.IAA is the most common in the auxins, and has been the subject of extensive studies by plant physiologists (Simen & Petrášek, 2011).IBA is a plant hormone in the auxin family and is an ingredient in many commercial horticultural plant rooting products (Ludwig-Müller, 2000).1-naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) are synthetic plant hormone in the auxin family and also used for plant tissue culture.NAA is a rooting agent and used for the vegetative propagation of plants from stem and leaf cutting (Naduvilpurakkal et al., 2014).2,4-D is one of the most widely used herbicides in the world.2,4-D and as a synthetic auxin it is often used in laboratories for plant research and as a supplement in plant cell culture media such as MS medium (Andrew & Reade, 2010).
Our previous studies indicated that as the young spike developed, endogenous IAA contents in branched spike wheat were much higher than those in normal spike lines.Meanwhile, the relative expression of TaIAR3, an auxin synthesis related gene, in branched spike wheat line was also significantly higher than those in normal spike lines.Higher IAA content might involve in the formation and growth of branched rachis in branched spike wheat (Data published lately).
LEAFY, or FLORICOULA (FLO)/LEAFY (LFY) genes were characterized initially as floral meristem genes in Antirrhinum and Arabidopsis (Coen et al., 1990;Weigel et al., 1992), which act as genetic switches during the choice of floral versus shoot fates (Yanofsky, 1995).Expression of FLO/LFY has been observed in young vegetative shoot apices and leaf primordia and might involve in the inflorescence architecture which distinct controlling the initiation of floral meristems in many plant species, including Oryza sativa (Kyozuka et al., 1998) and Z. mays (Bomblies et al., 2003).
The objective of the present study was to compare the effect of natural and synthetic auxins on the branched spike traits, growth and photosynthesis related characters, and the expression pattern of TFL in the shoot apex of plant was also determined in responses to auxins.The obtained results may be important for elucidation of the plant hormone role in branched spike development in wheat.

Plant Materials, Growth and Treatments
Fen 33, a released branched spike wheat cultivar in HuangHuan wheat zone in China, was grown in Agronomy Experimental Station of Shandong Agriculture University, Taian City, Shandong Province, China, from 2012 to 2013 growing season.The experiment plot was about 8.0 m 2 and the wheat plants were thinly sowed at 6 cm plant spacing and 22 cm row spacing on 8th October 2012.Cultivation management was similar with common practice at local place.Sowed wheat lines turned green at mid February in 2013.The young shoots were tripped out and observed under microscopy to determine the development stages of shoot apexes.Auxins dissolved in 50% ethanol were applied at experimental concentrations, 0.1 mM IBA, 0.1 mM IAA, 1.0 mM NAA and 1.0 mM 2,4-D (Sigma-Aldrich Co., USA), respectively.An equal amount of 50% ethanol was added to the control.Auxins treatments were performed three times on 3, 6 and 9 days after the two ridges stage of shoot apex.Plants were foliar sprayed until considerable run-off on the leaf surface occurred at early morning.The experiments were carried out with complete random block design with four replicates.

Determination of Pike Characters
After wheat maturation at June, twenty spikes of main shoots in each replicate plot were randomly taken out to investigate the number and the length of longest branched rachis, the number of spikelet and fertile floret.Numbers of branched rachis and spikelet were counted.The length of branched rachies was measured by using a meter scale.Fertile floret was represented as the grain number in a spike which was counted at the same time.

Determination of Growth Traits and Photosynthesis Related Characters
After 30 d of first auxins treatment, the plants were removed from the field along with the soils and were dipped in a bucket filled with water.The plants were moved smoothly to remove the adhering soils particles were weighted for fresh mass, and then placed in an oven run at 80 o C for 24 h.These dried plants were weighed to record the plant dry mass.Chlorophylls a, b and carotenoids contents (mg g -1 FW) were detected according to the methods given by Arnon (1949).Clean leaf materials were homogenized with 80% acetone and centrifuged; the optical density of the acetone extract was measured at 663, 645 and 470 nm using a UV-160A UV Visible Recording Spectrometer, Shimadzu, Japan.

Isolation of LFY/FLO Homologue TFL in Branched Spike Wheat
The strategy of direct sequencing of a reverse transcription PCR product was used to obtain the putative homologue of LFY/FLO, using degenerate primers and cDNA from shoot apical meristems of branched spike wheat plants expected to express a LFY/FLO homologue.Total RNA was isolated from young shoot apex tissues using TRIzol reagent (Invitrogen) and RT-PCR was performed using the degenerate forward primer 5′CGC(G)GAGCTC(G)GACGACATGA3′ and reverse primer 5′GCACTGCTCGTAG(C)AGA(G)TGGA3′ to obtain the middle region of TFL.The following thermocycling conditions were employed: initial denaturation at 94 o C for 3 min; 30 cycles of 94 o C for 30 s, 59 o C for 45 s, and 72 o C for 1 min; final extension at 72 o C for 10 min.The amplified products were separated on a 1.5% agarose gel, visualised and photographed.
The amplified cDNA fragment was independently cloned at least twice into pGM-T Easy vector (Promega) and sequenced.After sequencing, the sequence was submitted to GenBank to identify similar genes.To obtain the full length cDNA sequence of TFL, a set of specific primers were designed for the 5′ and 3′ RACEs (Rapid Amplification of cDNA Ends) by using the SMARTTM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA).These primers were: 5′Race: PF: 5′TTTCTTGGCCTTTCTCGCCA3′; 3′Race: PR: 5′AAGAAGAACGGGCTGGACTA3′; The amplified sequences were cloned and sequenced.The full length cDNA containing ORF for TFL was obtained by PCR with forward primer (5′ATGGATCCATGGATCGCCACGACGCCT3′) and reverse primer (5′TTCTAGCCTGTTGCCCGGAGGATCC3′).The primers contained the generated BamHI recognition site (GGATCC) to facilitate the cloning of the cDNAs.

Phylogenetic Analysis
Phylogenetic comparisons of amino acid sequences of different LFY/FLO homologues were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) and aligned with ClustalW mega (http://www.ebi.ac.uk/Tools/msa/clustalo/).Phylogenetic trees based on the complete sequences were generated using MEGA4 and constructed by the neighbor joining (NJ) method.Bootstrap values were derived from 1000 replicate runs.

Real-Time RT-PCR
For experiments on apices total RNA was extracted from the apical part of the plants.The samples were taken before the beginning of the auxins treatment and then every 10 d until the 30 th d after the beginning of the auxins first treatment.At first sampling the apical part of the plants included apex and a little of non-removable young shoots, and afterwards, only included the young spike.RNA was isolated TRIzol from Invitrogen following manufacturer's protocol.Each RNA sample was treated with RNase-Free.DNase (Promega) following manufacturer's protocol in an effort to remove any residual genomic DNA (gDNA).DNase treated RNA was subjected to reverse transcriptase reactions using oligo-dT primer and PrimeScriptTM Reverse Transcriptase (Takara) according to manufacturer's protocol.The gene-specific primers used in RT-PCR were 5′AGAACGACTGCGACGACGA3′ and 5′CCGCATCTTGGGCTTGTTGA3′.As a control, the cDNA sequence of the actin gene was amplified by using the two primers 5′CACGGCATCGTAAGCAACTG3′ and 5′TCCTTCGTAAATGGGCACGGT3′. The following thermocycling conditions were employed: initial denaturation at 94 o C for 3 min; 30 cycles of 94 o C for 30 s, 57 o C for 45 s, and 72 o C for 1 min; final extension at 72 o C for 7 min.The amplified products were separated on a 1.5% agarose gel, visualised and photographed.The obtained CT values were analyzed by averaging three independently calculated normalized expression values for each sample.PCR products were sequenced to ensure amplification products for the purpose of gene fragment.Expression values are given as the mean of the normalized expression values of the triplicates, calculated according to 2 -ΔΔCT method (Livak & Schmittgen, 2001).jas.ccsenet.

Results
The The 2.41-3.95%increase in chlorophyll a level and 1.12-8.40%increase in chlorophyll b were noted in response to 1.0 mM NAA and l.0 m 2,4-D.These auxins also stimulated the carotenoid content by 4.11-8.87%after 30 d of first treatment.

Photosynthesis Rate, Intercellular Concentration of Carbon Oxide, Stamatal Conductance and Transpiration Rate Determination
Wheat in the presence of 0.1 mM IBA had 13.3%, 36.1%,21.9% and 9.42% more photosynthesis rate, intercellular concentration of carbon oxide, stamatal conductance and transpiration rate, respectively, than the control after 30 d of first treatment (Table 1).A significant increase in the photosynthetic parameters (4.62% in the case of photosynthesis rate, 24.1% in the case of intercellular concentration of carbon oxide, 9.36% in the case of stamatal conductance and 7.01% in the case of transpiration rate) was also observed with 0.1 mM IAA application.In addition, exposure of branched wheat to 1.0 mM NAA or 2,4-D caused a weaker, but statistically significant increase in the photosynthesis rate (4.58-17.1%),intercellular concentration of carbon oxide (11.4-13.7%),stamatal conductance (6.25-15.6%)andtranspiration rate (1.83-6.67%)after 30 d of first treatment.

Expression of TFL during Spike Development
The effect of auxins on the expression of the TFL gene was examined (Genbank accession number: KP408434).The coding region of TFL is 1,185 bp, and encodes a putative protein of 394 amino acids, which is 59.2% identical to WFL (GenBank accession No: AB231888), a LFY/FLO homologous gene from the species of common wheat (Shitsukawa et al., 2006) Auxin plays an important role in the process of plant growth and development (Hohm et al., 2013).0.1 mM IBA were the most effective in promoting plant growth, and photosynthetic capability.The effects of synthetic auxins NAA and 2, 4-D were significantly lower than those of IBA and IAA.A significant increment in growth, pigment content, and activities of antioxidant enzymes could be noticed in wheat young seedlings when the seeds were pretreated with IAA (Agami & Mohamed, 2013).In Catharanthus roseus, IBA, IAA and NAA significantly improved plant fresh and dry weights, total chlorophyll and carotenoids content, and net photosynthetic rate (Alam et al., 2012).In Panax ginseng, that exogenous IAA enhanced significantly net photosynthetic rate, stomatal conductance, transpiration rate at all growth stages (Li & Xu, 2014).The results in this study suggest there were no negative effects of auxins on wheat growth and photosynthesis when more fertile floret obtained in branched spike wheat by auxins application.
Evidence provided in this study supports the suggestion that TFL is the branched spike wheat homologue of FLO/LFY.A high degree of similarity was seen between the isolated cDNA molecule and fragments of known FLO/LFY homologues.TFL expressed at a low level in the shoot apex of control and auxin-treated plants at the start of the experiment, showing that TFL was expressed at the end of two ridges stage of wheat, at this time the shoot apex did not enter into the stage of floral organogenesis.
In no auxins and auxins treated plants, the quantities of TFL unregulated at the time when spike were undergoing the branched rachises differentiated and elongation.That implicates TFL might involved in branched rachis transition and possibly in branched rachis organogenesis.
In flower plants, FLO/LFY encodes a plant-specific transcriptional factor (Blazquez et al., 1997).The primary function of FLO/LFY homologous genes is to repress the development of vegetative organs and to promote the formation of flower meristem and then also influence blossum time (Mandel et al., 1995;Wada et al., 2002;Molinero-Rosales et al., 1999).The other function of FLO/LFY homologous genes is to maintain the activity of flower meristem and to activate the genes that specify the development of different flower organs (Weigel et al., 1992;Carmona et al., 2002).
Previous studied documented that FLO/LFY homologs might involve in inflorescence architecture.In maize, zfl1 and zfl2 are duplicate FLO/LFY homologs in maize.Transposon insertions into the two maize genes led to a disruption of floral organ identity and patterning, as well as to defects in inflorescence architecture and in the vegetative to reproductive phase transition (Bomblies & Doebley, 2005).In normal spike wheat, the expression pattern of WFL, a FLO/LFY ortholog, indicated that WFL is associated with spikelet formation rather than floral meristem identity (Shitsukawa et al., 2006).Auxins induced increasement of the expression of TFL over this time, although the extent to which TFL levels increased were associated with the kinds of auxins.This indicates that TFL may be involved in the formation and branching, and its expression might be induced by auxins.

Conclusions
The four types of experienced auxins induced similar effects on various physiological processes, including growth traits, photosynthetic parameters, although the impact extents were different among them.The data supports the hypothesis that auxins play a central role in the regulation branched spike characters and TFL might correlate with the development of branched rachises in branched spike wheat.

Table 1 .
The effect of auxins (IAA, IBA, NAA, 2,4-D)on growth traits, pigment contents, and photosynthesis related characters in branched spike wheat after 30 days of first treatment.Data are the means of four independent experiments ± SD .1 mM had the most stimulatory effect on chlorophyll a, chlorophyll b and carotenoid accumulation after 30 d of first treatment.Other auxins were characterized by lower stimulatory effects on photosynthetic pigment levels in branched spike wheat.IAA at a concentration of 0.1 mM stimulated chlorophyll a accumulation by 7.69%, chlorophyll b by 32.2% and carotenoid by 4.54% after 30 d of first auxin treatment.