ФИЗИОЛОГИЯ РАСТЕНИЙ, 2015, том 62, № 5, с. 729-739
GENE EXPRESSION PROFILING IN SHOOT APICAL MERISTEM
OF Gossypium hirsutum1
© 2015 M. Wu*2, J. Li*2, S. L. Fan*, M. Z. Song*, C. Y. Pang*, J. H. Wei*, J. W. Yu*,
J. F. Zhang**, S. X. Yu*
*State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, Henan, China **Department of Plant and Environmental Sciences, New Mexico State University, Las Cruces, USA
Received August 8, 2014
Early maturity is a particularly important agronomic trait for cotton breeding in China and is determined by many morphological and phenological traits. The time of floral initiation is one of most important factors related to early maturation of cotton. The aim of this study was to identify differentially expressed (DE) genes related to floral initiation using the Arabidopsis thaliana GeneChip® system on the shoot apical meristems (SAMs) of an early-maturing cotton cultivar. Compared with SAMs at 10 days, 365 genes were differentially expressed in the SAMs at 20 days. Of these, 210 and 155 transcripts were up- and down- regulated, respectively. The results of Gene Ontology (GO) annotation indicated that most genes fell into the four largest functional groups: metabolism, transposable elements, protein binding or cofactor requirements, and protein fate. These groups constituted 18.9, 11.8, 12.8 and 6.8% of the total DE genes, respectively. Many DE genes were identified, including those encoding the transcription factors SOC1-like floral activator MADS4, B3-domain containing transcription factor, MYB2, MYB85, and GHMADS-1. Our research found that the B3-domain containing transcription factor was similar to Arabidopsis genes encoding auxin response factor 36 and VERNALIZATION 1 (VRN1) and was one of 155 down-regulated 'Apex-unique' transcripts. The B3-domain containing transcription factor was 1128 bp long and was named GhV1 (GenBank accession No. GU929695). The induction of the transcripts we identified in the cotton SAM after 10 days of SD revealed that the transition to reproductive development occurred at this particular time point. These results allowed for a detailed description of temporal gene expression changes in the cotton SAM as it undergoes the floral initiation process.
Keywords: Gossypium hirsutum — maturity — timing of floral transition — gene chip — differentially expressed genes — GhV1 gene
Cotton (Gossypium hirsutum) is an economically important fiber crop. Early maturity in cotton is a particularly important agronomic trait for cotton breeding in China and is determined by many interconnected morphological and phenological traits. These traits include days from planting to floral initiation, squaring, flowering and open bolls, boll maturation period, and percentage of yield at early harvest or before frost . Because squares that develop into flowers arise from fruit-
1 This text was submitted by the authors in English.
2 These authors contributed equally to the work.
Abbreviations'. DE — differentially expressed; FAA — formalin acetate alcohol; SD — short-day length; SAMs — shoot apical meristems; VRN1 — reduced vernalization response 1; NCBI — National Center for Biotechnology Information. Corresponding authors. Yu Shu-Xun, Zhang Jin-Fa, and Yu Ji-Wen. State Key Laboratory of Cotton Biology, Institute of Cotton Research of CAAS, Anyang, Henan, 455000, China; fax. +86 372 2562275; e-mails. firstname.lastname@example.org; email@example.com; firstname.lastname@example.org
ing branches (sympodia) that appear after vegetative branches (monopodia) from the lower main-stem nodes, the first fruiting branch is an easily observable marker of days to floral initiation (usually at the 2nd to 3rd true leaf stage) and squaring.
In recent years, genetic studies have revealed dozens of genes involved in the floral transition [2—4]. Schmid et al.  used an excised shoot apex system consisting of the shoot apical meristem (SAM) with leaf primordia and developing leaves to study gene expression changes at different time points following floral induction in Arabidopsis. In maize SAMs, up-regulated expressed sequence tags (ESTs) involved many regulatory genes, including transcription factors, chromatin remodeling factors, and components of the gene-silencing machinery, along with approximately 900 genes of unknown function . In soybean, a total of 331 transcripts displayed significant changes in their expression profiles. These transcripts included genes involved in sugar, auxin, or abscisic acid (ABA) regulation in events prior to the induction of floral ho-
WU h gp.
meotic transcripts . In poplar (Populus tremula x x Populus alba), a large set of genes commonly expressed during growth-to-dormancy transitions was identified in apical buds . Comparison with genes induced during floral induction of poplar, maize, soybean or Arabidopsis suggested that the molecular mechanisms underlying such transitions were similar in different plant organs.
Microarray technology is a powerful tool for analyzing the expression of thousands of genes in a single experiment [9, 10]. Affymetrix GeneChips have set the standard in terms of reproducibility, sensitivity and other quality parameters relative to other oligonucleotide or cDNA array platforms . Unfortunately, plant-related Affymetrix Whole Genomic GeneChips are only available for a limited number of plant species. Although GeneChip Cotton arrays are also available, they have been used infrequently in the research community due to the limited genome coverage offered and the numerous ESTs that are not annotated.
One possible solution to this limitation is to utilize heterologous microarrays from related species . For example, heterologous Arabidopsis GeneChips have been recently used to study drought stress in a non-model banana, Musa spp. . Boutanaev et al. reported the successful use of heterologous microar-rays, including those for Arabidopsis and rice, for the study of defense responses in plants . Horvath et al. used an Arabidopsis cDNA microarray to identify conserved and differentially expressed (DE) genes involved in shoot growth and development in distantly related plant species, such as Arabidopsis leafy spurge, poplar, and wild oat . Tomato microarrays were used to compare gene expression among several Solanaceae species, including pepper, eggplant, and potato [16, 17]. Similarly, Arabidopsis appears to be compatible with cotton for conducting a heterologous microarray approach because the two species are phy-logenetically related . Carica papaya, Gossypium raimondii, Theobroma cacao and Arabidopsis thaliana belong to a subclade that diverged approximately 82.3 million years ago . A cotton cDNA microar-ray was used to identify changes in gene expression in velvetleaf due to competition with corn . Nazar et al. recently demonstrated that "whether using homologous or heterologous arrays, the chances of seeing interesting differences are similar" . Therefore, heterologous microarrays can be used to study gene expression before complete genome sequence data are available for even distantly related species of interest.
In the present study, gene expression profiling was designed to analyze genes which expression patterns were altered between short-season cotton and later-maturing genotypes. We used the Arabidopsis GeneChip microarray to query the cotton SAM as it underwent the floral initiation process.
MATERIALS AND METHODS
Plant materials and growth conditions. Plants of CCRI36 (a short-season cotton cultivar with a whole growth period of 107 days) and TM-1 (a later-maturing genotype with a whole growth period of 132 days) were grown under standard field conditions in Anyang, Henan, China during the summer of 2009. These two cotton genotypes were sown at the same time under the same management conditions. After seeds germinated, we carefully observed and recorded the emergence time, leaf unfolding and rate of growth. From the cotyledon to the first true leaf stage, seedling shoot apices were collected for histology and fixed in FAA (formalin-acetate-alcohol). Shoot apices starting at the base of the apex and any leaf primordia <0.2 mm in length (after exposure to 10 and 20 days of short-day length, SD), mature floral buds (bracts, sepals, petals, stamens, and carpels), roots, stems, fiber, ovules, and leaves of the CCRI36 cultivar were prepared, frozen in liquid nitrogen, and stored at —80°C until further processing.
Histological techniques and microscopy. Under our experimental conditions, plants were grown under SD conditions, and SAMs were dissected after exposure to 5 to 40 days of SD in order to capture transcripts that were essential in establishing the floral meristem. Apices were harvested in triplicate and used for dissection and production of paraffin sections.
RNA preparation. RNA isolation was conducted using the Sigma Spectrum™ Plant Total RNA Kit ("Sigma-Aldrich", USA) with on-column DNasel digestion according to the manufacturer's instructions. RNA quantity was determined by using a DU®800 spectrophotometer ("Beckman coulter", USA). An RNA integrity number (RIN) was determined for each sample. Only samples with RIN values of 1.9 to 2.1 were used for further analysis. An oligo(dT) primer was used to prime first-strand cDNA synthesis from 5 ^g of total RNA using the SuperScript III reverse transcriptase enzyme ("Invitrogen", USA) according to the manufacturer's instructions. A single reverse transcription (RT) reaction was performed for each RNA sample.
Microarray experiments and statistical analysis. To
analyze the global pattern of gene expression in the cotton SAMs, microarray experiments with three technical replicates and three biological replicates were performed using shoot apices after exposure to 10 and 20 days of SD. Microarray experiments were perfor
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