Sucrose accumulation and the expression of neutral invertase in sugarcane
Date
2001-12
Authors
Rose, Susan, 1977-
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: The goals of this project were to (i) determine maximum extractable neutral invertase (NI)
activity in the sugarcane culm, (ii) sequence a cDNA encoding for the sugarcane NI (SNI),
(iii) determine SNI copy number in the genome, (iv) describe SNI transcript and protein
expression patterns throughout the plant, and (v) attempt to determine the contribution of
hydrolysis to sucrose accumulation.
SNI and sugars were extracted from the developing culm tissues of sugarcane,
commercial variety N19. Tissues were divided according to developmental stage
(internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular or
storage tissues). The lowest sucrose content was found in the core of the bottom of each
of the internodes. The ratio between hexoses and sucrose was highest in the young
internodes. In these internodes hexose content was higher in the bottom than the top.
There was a significant correlation between sucrose content and NI. Fluxes involved in
sucrose synthesis and hydrolysis were investigated. The hexoses glucose and fructose
were supplied as a carbon source for tissue discs of young and maturing internodal tissues
of sugarcane, varieties N19 and US6656-15. Sucrose content was 10-fold higher in
maturing internodes of N19 than US6656-15. Calculated sucrose hydrolysis rates via
invertase were higher in maturing internodes of US6656-15 than N19. Taking metabolic
compartmentation into account, hydrolysis of sucrose via invertase made a significant
contribution to the net turnover of sucrose. Along with this, it would appear that the ability
to partition sucrose between the vacuole and cytosol causes a significant difference in
sucrose content between varieties.
A full-length cDNA for SNI was sequenced. This expressed gene showed significant
homology to known NI sequences on both nucleic and amino acid levels. The SNI
sequence did not contain the putative invertase catalytic amino acid sequence, suggesting
it developed separately from the other classes of invertases. Approximately 1.8 kb of the
SNI cDNA was incorporated into a vector suitable for direct bombardment into sugarcane
tissue. Southern blot analysis showed the enzyme has a low copy number. SNI transcript
expression was observed in all tissues of the sugarcane plant: roots, internodes, leaf roll
and leaves. In culm tissues where sucrose content was low and hexose contents were
high, SNI transcript and protein levels were high. This suggests that SNI is involved in
growth metabolism.
AFRIKAANSE OPSOMMING: Die doel van die projek was om (i) maksimum ekstaheerbare neutrale invertase (NI) aktiwiteit in die suikerriet stingel te bepaal, (ii) die volgorde van 'n eDNA wat vir suikerriet NI (SNI) kodeer te bepaal, (iii) die SNI kopie-getal in die genoom te bepaal, (iv) SNI m- RNA en proteïenuitdrukkingspatrone deur die plant te beskryf, en (v) te poog om die bydrae van hidrolise op sukrose akkumulering te bepaal. SNI en suikers is geëkstraheer uit 'n kommersiële varieteit, N19. Weefsels was volgens ontwikkelingstadiums (internodes 3, 6 en 9) en anatomiese verskille (verryking vir groeiende, vaat- en bergings-weefsels) verdeel. Die laagste sukrose inhoud is in die middel van die onderste helfte van elke internode gevind. Die verhouding van heksoses tot sukrose was die hoogste in die jong internodes. Die inhoud heksoses was hoër in die onderste deel van die internode as die boonste deel. 'n Betekenisvolle korrelasie tussen sukrose inhoud en SNI is gevind. Flukse betrokke by sukrose sintese en hidrolise is ondersoek. Glukose en fruktose is as koolstofbron aan stingelweefsel van twee variëteite (US6656-15 and N19) toegedien. Sukrose-inhoud het tienvoudig tussen volwasse weefsels van die twee variëteite verskil. Hidrolise via invertase was hoër in ouer weefsels van US6656-15 as N19, en het In noemenswaardige bydrae tot sukroseomset gemaak. Die verdeling van sukrose tussen die vakuool en die sitosol kan moontlik 'n groot rol speel in die vermoë van die sel om sukrose te akkumuleer. Die volgorde van 'n volledige SNI eDNA is bepaal. The uitgedrukte geen het, op beide In nukleïen- en aminosuur vlak, betekenisvolle ooreenkoms getoon met ander bekende plant NI volgordes. Die SNI volgorde bevat nie die kenmerkende invertase katalitiese setel nie, wat daarop kan dui dat dit onafhanklik van ander klasse invertases ontwikkel het. Min of meer 1.8 kb van die SNI eDNA is in 'n vektor geskik vir bioliestiese transformering van suikerrietweefsel, geïnkorporeer. Southern klad analise het gewys dat die ensiem 'n lae kopiegetal op geen vlak het. SNI mRNA uitdrukking is waargeneem in elke weefseltipe van die suikerriet plant: wortels, internodes, blaarrol en blare. In stingelweefsels met lae sukrose- en hoë heksose-inhoud, was die vlakke van beide SNI-mRNA en -proteïen hoog. Dit dui daarop dat SNI moontlik betrokke is by groei-metabolisme.
AFRIKAANSE OPSOMMING: Die doel van die projek was om (i) maksimum ekstaheerbare neutrale invertase (NI) aktiwiteit in die suikerriet stingel te bepaal, (ii) die volgorde van 'n eDNA wat vir suikerriet NI (SNI) kodeer te bepaal, (iii) die SNI kopie-getal in die genoom te bepaal, (iv) SNI m- RNA en proteïenuitdrukkingspatrone deur die plant te beskryf, en (v) te poog om die bydrae van hidrolise op sukrose akkumulering te bepaal. SNI en suikers is geëkstraheer uit 'n kommersiële varieteit, N19. Weefsels was volgens ontwikkelingstadiums (internodes 3, 6 en 9) en anatomiese verskille (verryking vir groeiende, vaat- en bergings-weefsels) verdeel. Die laagste sukrose inhoud is in die middel van die onderste helfte van elke internode gevind. Die verhouding van heksoses tot sukrose was die hoogste in die jong internodes. Die inhoud heksoses was hoër in die onderste deel van die internode as die boonste deel. 'n Betekenisvolle korrelasie tussen sukrose inhoud en SNI is gevind. Flukse betrokke by sukrose sintese en hidrolise is ondersoek. Glukose en fruktose is as koolstofbron aan stingelweefsel van twee variëteite (US6656-15 and N19) toegedien. Sukrose-inhoud het tienvoudig tussen volwasse weefsels van die twee variëteite verskil. Hidrolise via invertase was hoër in ouer weefsels van US6656-15 as N19, en het In noemenswaardige bydrae tot sukroseomset gemaak. Die verdeling van sukrose tussen die vakuool en die sitosol kan moontlik 'n groot rol speel in die vermoë van die sel om sukrose te akkumuleer. Die volgorde van 'n volledige SNI eDNA is bepaal. The uitgedrukte geen het, op beide In nukleïen- en aminosuur vlak, betekenisvolle ooreenkoms getoon met ander bekende plant NI volgordes. Die SNI volgorde bevat nie die kenmerkende invertase katalitiese setel nie, wat daarop kan dui dat dit onafhanklik van ander klasse invertases ontwikkel het. Min of meer 1.8 kb van die SNI eDNA is in 'n vektor geskik vir bioliestiese transformering van suikerrietweefsel, geïnkorporeer. Southern klad analise het gewys dat die ensiem 'n lae kopiegetal op geen vlak het. SNI mRNA uitdrukking is waargeneem in elke weefseltipe van die suikerriet plant: wortels, internodes, blaarrol en blare. In stingelweefsels met lae sukrose- en hoë heksose-inhoud, was die vlakke van beide SNI-mRNA en -proteïen hoog. Dit dui daarop dat SNI moontlik betrokke is by groei-metabolisme.
Description
Thesis (MSc)--University of Stellenbosch, 2001.
Keywords
Sugarcane -- Genetics, Invertase, Sucrose, Hydrolysis