Mi Zhang

spatiotemporal manipulation of auxin biosynthesis in cotton ovule epidermal cells enhances fiber yield and quality

The capacity of conventional breeding to simultaneously improve the yield and quality of cotton fiber is limited. The accumulation of the plant hormone indole-3-acetic acid (IAA) in cotton fiber initials prompted us to investigate the effects of genetically engineering increased IAA levels in the ovule epidermis. Targeted expression of the IAA biosynthetic gene iaaM, driven by the promoter of the petunia MADS box gene Floral Binding protein 7 (FBP7), increased IAA levels in the epidermis of cotton ovules at the fiber initiation stage. This substantially increased the number of lint fibers, an effect that was confirmed in a 4-year field trial. The lint percentage of the transgenic cotton, an important component of fiber yield, was consistently higher in our transgenic plants than in nontransgenic controls, resulting in a >15% increase in lint yield. Fiber fineness was also notably improved.

Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis

Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.

Molecular Cloning and Characterization of a Cytokinin Dehydrogenase Gene from Upland Cotton (Gossypium hirsutum L.)

Cytokinins are plant hormones that play crucial roles in plant growth and development. Cytokinin dehydrogenase (CKX), regarded as a main negative regulator in cytokinin metabolism in plants, irreversibly degrades cytokinins into adenine/adenosine moiety. A CKX homologous gene, designated GhCKX, was cloned from upland cotton (Gossypium hirsutum L.). Transgenic tobacco plants over-expressing GhCKX showed a typical cytokinin-deficient phenotype, while CKX-silenced tobacco plants exhibited cytokinin over-producing phenotype. Tissue specifically enhancing the expression of GhCKX in the ovule epidermis of transgenic cotton led to a significant decrease of trans-zeatin and trans-zeatin riboside contents in the ovule. The decline of cytokinins resulted in a significant decrease in fiber initials on a single ovule. Our results indicate that GhCKX encodes a functional CKX, and cytokinins may be required for the initiation of cotton fiber cells.

Transcriptome and Biochemical Analyses Revealed a Detailed Proanthocyanidin Biosynthesis Pathway in Brown Cotton Fiber

Brown cotton fiber is the major raw material for colored cotton industry. Previous studies have showed that the brown pigments in cotton fiber belong to proanthocyanidins (PAs). To clarify the details of PA biosynthesis pathway in brown cotton fiber, gene expression profiles in developing brown and white fibers were compared via digital gene expression profiling and qRT-PCR. Compared to white cotton fiber, all steps from phenylalanine to PA monomers (flavan-3-ols) were significantly up-regulated in brown fiber. Liquid chromatography mass spectrometry analyses showed that most of free flavan-3-ols in brown fiber were in 2, 3-trans form (gallocatechin and catechin), and the main units of polymeric PAs were trihydroxylated on B ring. Consistent with monomeric composition, the transcript levels of flavonoid 3′, 5′-hydroxylase and leucoanthocyanidin reductase in cotton fiber were much higher than their competing enzymes acting on the same substrates (dihydroflavonol 4-reductase and anthocyanidin synthase, respectively). Taken together, our data revealed a detailed PA biosynthesis pathway wholly activated in brown cotton fiber, and demonstrated that flavonoid 3′, 5′-hydroxylase and leucoanthocyanidin reductase represented the primary flow of PA biosynthesis in cotton fiber.

Auxin Regulates Cotton Fiber Initiation via GhPIN-Mediated Auxin Transport

Cotton fibers are seed trichomes that make cotton unique compared with other plants. At anthesis, IAA, a major auxin in plants, accumulates in the fiber cell to promote cell initiation. However, many important aspects of this process are not clear. Here, auxin distribution patterns indicated by auxin-dependent DR5::GUS (β-glucuronidase) expression in cotton ovules were studied during fiber cell differentiation and cell initiation [-2 to 2 DPA (days post-anthesis)]. The nucellus and fiber cell were two major sites where auxin accumulates. The accumulation in the nucellus started from -1 DPA, and that in fiber cells from 0 DPA. Immunolocalization analysis further suggests that the IAA accumulation in fiber initials began before flower opening. Furthermore, we demonstrate that accumulated IAA in fiber initials was mainly from efflux transport and not from in situ synthesis. Eleven auxin efflux carrier (GhPIN) genes were identified, and their expression during ovule and fiber development was investigated. Ovule-specific suppression of multiple GhPIN genes in transgenic cotton inhibited both fiber initiation and elongation. In 0 DPA ovules, GhPIN3a, unlike other GhPIN genes, showed additional localization of the transcript in the outer integument. Collectively, these results demonstrate the important role of GhPIN-mediated auxin transport in fiber-specific auxin accumulation for fiber initiation.

PIN-formed protein, a door to reveal the mechanism for auxin-triggered initiation of cotton fiber

ABSTRACT Cotton fibers are differentiated ovule epidermal cells that provide an ideal model to study cell differentiation and elongation. Establishment of auxin maximum in fiber cells is crucial for cotton-fiber protrusion from ovule surface. However, it is unclear where the auxin originates from and how the auxin accumulates in fiber cells. Our recent results indicate that the auxin is mainly imported from the outside of ovules, and transported to fiber cells through GhPIN (homolog of PIN-formed proteins in cotton) -mediated polar auxin transport, rather than in situ synthesis. Based on our finding in GhPINs, we discuss here briefly how auxin flow to fiber cells and auxin gradient in ovule epidermis is established mainly by GhPIN3a protein.

In vivo imaging of Ca2+ accumulation during cotton fiber initiation using fluorescent indicator YC3.60

Key messageNon-tip-focused Ca2+gradient indicated by genetically expressing a FRET-based calcium sensor YC3.60 was established in spherical expanding cotton fibers, which is vital for cotton fiber initiation.AbstractCotton fiber is a single cell elongated from ovule epidermis. It is not only the most important natural fiber used in the textile industry but also an ideal model for studying cell differentiation and elongation. Before linear cell growth, cotton fibers undergo spherical expansion at the beginning of initiation. Ca2+, as an important secondary messenger, plays a central role in polarized cell growth including cotton fiber elongation. However, the role of Ca2+ in fiber initiation is far from well understood. In this paper, through ovule culture we demonstrate that Ca2+ is crucial for fiber initiation. Using transgenic cotton expressing the fluorescent Ca2+ indicator YC3.60, we show cellular and intracellular distribution of Ca2+ in cotton ovule epidermis and fiber cells. In the initiating fiber cell, Ca2+ accumulated mainly at the base of the cell, while in the fast elongating cell, the Ca2+ was enriched in the tip region. This cellular distribution of Ca2+ reported by YC3.60 was confirmed by the staining with a Ca2+-sensitive dye fluo-3/AM. Compared to the fluorescent dye staining, the YC3.60 system can reveal more detailed information on the intracellular distribution without photobleaching. Taken together, our data suggest that Ca2+ plays an important role in spherical expansion of cotton fiber initials.