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RNA-seq analysis of short fiber mutants Ligon-lintless-1 (Li 1 ) and - 2 (Li 2 ) revealed important role of aquaporins in cotton (Gossypium hirsutum L.) fiber elongation.

Naoumkina M, Thyssen GN, Fang DD - BMC Plant Biol. (2015)

Bottom Line: We found a large number of differentially expressed genes common to both mutants, including 531 up-regulated genes and 652 down-regulated genes.We found that the aquaporins were the most down-regulated gene family in both short fiber mutants.These results suggest that higher accumulation of ions in fiber cells, reduced osmotic pressure and low expression of aquaporins, may contribute to the cessation of fiber elongation in Li 1 and Li 2 short-fiber mutants.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cotton fiber length is a key determinant of fiber quality for the textile industry. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon lintless-1 (Li 1 ) and Ligon lintless-2 (Li 2 ) are monogenic and dominant mutations, that result in an extreme reduction in the length of lint fiber to approximately 6 mm on mature seeds. In a near-isogenic state with wild type (WT) cotton these two short fiber mutants provide an excellent model system to study mechanisms of fiber elongation.

Results: We used next generation sequencing (RNA-seq) to identify common fiber elongation related genes in developing fibers of Li 1 and Li 2 mutants growing in the field and a greenhouse. We found a large number of differentially expressed genes common to both mutants, including 531 up-regulated genes and 652 down-regulated genes. Major intrinsic proteins or aquaporins were one of the most significantly over-represented gene families among common down-regulated genes in Li 1 and Li 2 fibers. The members of three subfamilies of aquaporins, including plasma membrane intrinsic proteins, tonoplast intrinsic proteins and NOD26-like intrinsic proteins were down-regulated in short fiber mutants. The osmotic concentration and the concentrations of soluble sugars were lower in fiber cells of both short fiber mutants than in WT, whereas the concentrations of K+ and malic acid were significantly higher in mutants during rapid cell elongation.

Conclusions: We found that the aquaporins were the most down-regulated gene family in both short fiber mutants. The osmolality and concentrations of soluble sugars were less in saps of Li 1 - Li 2 , whereas the concentrations of malic acid, K+ and other detected ions were significantly higher in saps of mutants than in WT. These results suggest that higher accumulation of ions in fiber cells, reduced osmotic pressure and low expression of aquaporins, may contribute to the cessation of fiber elongation in Li 1 and Li 2 short-fiber mutants. The research presented here provides new insights into osmoregulation of short fiber mutants and the role of aquaporins in cotton fiber elongation.

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RNA-seq and RT-qPCR analyses of transcript level of members of the aquaporin family inLi1,Li2and WT developing fibers at 8 DPA. Error bars indicate standard deviation from 2 biological replicates for RNA-seq data and 3 biological replicates for RT-qPCR. Abbreviations: F, field grown plants; GH, greenhouse grown plants; PIP, plasma membrane intrinsic proteins; and TIP, tonoplast intrinsic proteins.
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Fig3: RNA-seq and RT-qPCR analyses of transcript level of members of the aquaporin family inLi1,Li2and WT developing fibers at 8 DPA. Error bars indicate standard deviation from 2 biological replicates for RNA-seq data and 3 biological replicates for RT-qPCR. Abbreviations: F, field grown plants; GH, greenhouse grown plants; PIP, plasma membrane intrinsic proteins; and TIP, tonoplast intrinsic proteins.

Mentions: Major intrinsic proteins or aquaporins were one of the most significantly (p < 0.0001) over-represented gene family among down-regulated genes in Li1 – Li2 fibers. Aquaporins facilitate the efficient transport of water and other small molecules across membranes in plants and other organisms [22]. Cotton aquaporins form a large family of proteins phylogenetically divided into five subfamilies including: plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently identified X (or unrecognized) intrinsic proteins (XIP) [23]. To assess which subfamily members of aquaporins were affected by Li1 – Li2 mutations: first, we conducted phylogenetic analysis of G. raimondii genes annotated as aquaporins; and second, evaluated their expression level in Li1 – Li2 developing fibers. The analyzed G. raimondii aquaporins clustered into five main clades (marked by empty squares) representing the above mentioned subfamilies (Additional file 3). The members of subfamilies PIP (7 genes), TIP (4 genes) and NIP (2 genes) were down-regulated in Li1 – Li2 developing fibers (marked by black triangle in Additional file 3). The most highly induced aquaporins in WT fibers, for which transcript levels were dramatically reduced in Li1 – Li2 mutants, were tested by RT-qPCR. In most cases results of RT-qPCR analysis were consistent with results of RNA-seq analysis (Figure 3). There were a number of aquaporins which showed increased transcript level only in greenhouse grown Li2 (Additional file 4), indicating interactive response to Li2 mutation and growth conditions. However, relative expression level of those genes was considerably less compared with WT expressed aquaporins as shown in Figure 3 (1,500 reads in greenhouse Li2 induced vs. 500,000 reads in WT expressed).Figure 3


RNA-seq analysis of short fiber mutants Ligon-lintless-1 (Li 1 ) and - 2 (Li 2 ) revealed important role of aquaporins in cotton (Gossypium hirsutum L.) fiber elongation.

Naoumkina M, Thyssen GN, Fang DD - BMC Plant Biol. (2015)

RNA-seq and RT-qPCR analyses of transcript level of members of the aquaporin family inLi1,Li2and WT developing fibers at 8 DPA. Error bars indicate standard deviation from 2 biological replicates for RNA-seq data and 3 biological replicates for RT-qPCR. Abbreviations: F, field grown plants; GH, greenhouse grown plants; PIP, plasma membrane intrinsic proteins; and TIP, tonoplast intrinsic proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4352256&req=5

Fig3: RNA-seq and RT-qPCR analyses of transcript level of members of the aquaporin family inLi1,Li2and WT developing fibers at 8 DPA. Error bars indicate standard deviation from 2 biological replicates for RNA-seq data and 3 biological replicates for RT-qPCR. Abbreviations: F, field grown plants; GH, greenhouse grown plants; PIP, plasma membrane intrinsic proteins; and TIP, tonoplast intrinsic proteins.
Mentions: Major intrinsic proteins or aquaporins were one of the most significantly (p < 0.0001) over-represented gene family among down-regulated genes in Li1 – Li2 fibers. Aquaporins facilitate the efficient transport of water and other small molecules across membranes in plants and other organisms [22]. Cotton aquaporins form a large family of proteins phylogenetically divided into five subfamilies including: plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently identified X (or unrecognized) intrinsic proteins (XIP) [23]. To assess which subfamily members of aquaporins were affected by Li1 – Li2 mutations: first, we conducted phylogenetic analysis of G. raimondii genes annotated as aquaporins; and second, evaluated their expression level in Li1 – Li2 developing fibers. The analyzed G. raimondii aquaporins clustered into five main clades (marked by empty squares) representing the above mentioned subfamilies (Additional file 3). The members of subfamilies PIP (7 genes), TIP (4 genes) and NIP (2 genes) were down-regulated in Li1 – Li2 developing fibers (marked by black triangle in Additional file 3). The most highly induced aquaporins in WT fibers, for which transcript levels were dramatically reduced in Li1 – Li2 mutants, were tested by RT-qPCR. In most cases results of RT-qPCR analysis were consistent with results of RNA-seq analysis (Figure 3). There were a number of aquaporins which showed increased transcript level only in greenhouse grown Li2 (Additional file 4), indicating interactive response to Li2 mutation and growth conditions. However, relative expression level of those genes was considerably less compared with WT expressed aquaporins as shown in Figure 3 (1,500 reads in greenhouse Li2 induced vs. 500,000 reads in WT expressed).Figure 3

Bottom Line: We found a large number of differentially expressed genes common to both mutants, including 531 up-regulated genes and 652 down-regulated genes.We found that the aquaporins were the most down-regulated gene family in both short fiber mutants.These results suggest that higher accumulation of ions in fiber cells, reduced osmotic pressure and low expression of aquaporins, may contribute to the cessation of fiber elongation in Li 1 and Li 2 short-fiber mutants.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Cotton fiber length is a key determinant of fiber quality for the textile industry. Understanding the molecular basis of fiber elongation would provide a means for improvement of fiber length. Ligon lintless-1 (Li 1 ) and Ligon lintless-2 (Li 2 ) are monogenic and dominant mutations, that result in an extreme reduction in the length of lint fiber to approximately 6 mm on mature seeds. In a near-isogenic state with wild type (WT) cotton these two short fiber mutants provide an excellent model system to study mechanisms of fiber elongation.

Results: We used next generation sequencing (RNA-seq) to identify common fiber elongation related genes in developing fibers of Li 1 and Li 2 mutants growing in the field and a greenhouse. We found a large number of differentially expressed genes common to both mutants, including 531 up-regulated genes and 652 down-regulated genes. Major intrinsic proteins or aquaporins were one of the most significantly over-represented gene families among common down-regulated genes in Li 1 and Li 2 fibers. The members of three subfamilies of aquaporins, including plasma membrane intrinsic proteins, tonoplast intrinsic proteins and NOD26-like intrinsic proteins were down-regulated in short fiber mutants. The osmotic concentration and the concentrations of soluble sugars were lower in fiber cells of both short fiber mutants than in WT, whereas the concentrations of K+ and malic acid were significantly higher in mutants during rapid cell elongation.

Conclusions: We found that the aquaporins were the most down-regulated gene family in both short fiber mutants. The osmolality and concentrations of soluble sugars were less in saps of Li 1 - Li 2 , whereas the concentrations of malic acid, K+ and other detected ions were significantly higher in saps of mutants than in WT. These results suggest that higher accumulation of ions in fiber cells, reduced osmotic pressure and low expression of aquaporins, may contribute to the cessation of fiber elongation in Li 1 and Li 2 short-fiber mutants. The research presented here provides new insights into osmoregulation of short fiber mutants and the role of aquaporins in cotton fiber elongation.

Show MeSH