FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA VEGETAL
PHOTOSYNTHESIS AND PHOTORESPIRATION IN THREE
C
4GRASSES OF DIFFERENT METABOLIC SUB-TYPES,
UNDER WATER STRESS
Ana Elizabete do Carmo Silva
DOUTORAMENTO EM BIOLOGIA
(Fisiologia e Bioquímica)
FACULDADE DE CIÊNCIAS
DEPARTAMENTO DE BIOLOGIA VEGETAL
PHOTOSYNTHESIS AND PHOTORESPIRATION IN THREE
C
4GRASSES OF DIFFERENT METABOLIC SUB-TYPES,
UNDER WATER STRESS
Ana Elizabete do Carmo Silva
DOUTORAMENTO EM BIOLOGIA
(Fisiologia e Bioquímica)
Tese orientada pela Professora Doutora Maria Celeste Arrabaça
The experimental work leading to this PhD was done in collaboration
with
ROTHAMSTED RESEARCH
Department of Plant Sciences
Under the supervision of Professor Dr. Martin A. J. Parry
And close advice of Dr. Alfred J. Keys
In addition to the supervision and orientation of Professor Celeste Arrabaça, Professor Martin Parry and Dr. Alfred Keys, the work here presented involved the collaboration with other Professors and Researchers. The four chapters of results contained in the Thesis (II, III, IV and V) correspond to the integral copy or extended versions of manuscripts submitted (or in final phase of preparation to be submitted) for publication in peer-reviewed journals. The relative contributions of each author apart from myself and the supervisors and advisors, who were involved in the planning of the experiments, discussion of the results and elaboration of manuscripts, are mentioned in the beginning of each chapter.
Para os devidos efeitos no n.º 2 do Art.8º do Decreto-Lei 388/70, o autor da tese declara que participou na execução do trabalho experimental descrito, bem como na análise e interpretação dos resultados e na redação dos textos e manuscritos submetidos para publicação.
Ana Elizabete do Carmo Silva Março de 2008
ACKNOWLEDGEMENTS / AGRADECIMENTOS
I am grateful to Fundação para a Ciência e a Tecnologia (Portugal) for financial support (PhD grant SFRH/BD/13730/2003), and to Faculdade de Ciências da Universidade de Lisboa (Portugal) and Rothamsted Research (UK) for receiving me and providing the conditions to do the work here presented. The grass seeds used were kindly provided by AgResearch, Margot Forde Forage Germplasm Centre, New Zealand and Alípio Dias & Irmão, Lda., Portugal.
Many people have somehow contributed to the successful realization of this PhD – many thanks to all!!! Here I can only express a special acknowledgement to a few of them!... Muitos foram aqueles que de alguma forma, directa ou indirectamente, contribuíram para a realização deste Doutoramento a bom termo – a todos, um grande bem-haja! Aqui, posso apenas agradecer a apenas algumas dessas pessoas!
Professora Doutora Maria Celeste Arrabaça pela sugestão do tema deste trabalho, pelo incentivo à sua execução e pelo acolhimento no Centro de Engenharia Biológica da FCUL. Agradeço-lhe a orientação e supervisão do trabalho, as demais lições científicas e afins, incluindo a da preserverança e a importância de manter a calma, e a disponibilidade que sempre mostrou para discutir os mais diversos aspectos que surgiram ao longo da sua realização. Agradeço-lhe também toda a amizade e o carinho.
Professor Dr. Martin Parry for receiving me at Rothamsted Research and showing me other faces of Science. I have learnt so many lessons while there, I have grown so much! I will always be thankful for the support, for the pushes applied when needed and for all the words and lessons – it did make a difference!
Dr. Alfred Keys, Alf… How can I find words that express the gratefulness for the many things you did for me? You were the main reason that made me to go to the UK and I will always owe you so much! The support, care and friendship, all the advices and the scientific or life-related discussions, the tears and the smiles… It is hard to imagine life far from you but (like with other parental relations) children must have wings and use them to fly around the world!
Todos os demais Professores e colegas do Departamento de Biologia Vegetal e do Centro de Engenharia Biológica da FCUL! Nomeadamente (e jamais exclusivamente!)... Prof. João Arrabaça pelo grande carinho (às vezes bem disfarçado!) com que me acolheu, pelo despertar do interesse pela cultura e pela transmissão de tão diversos e surpreendentes conhecimentos à “joven cientista”! Prof. Anabela Bernardes da Silva, Belucha, por me ter puxado para a Fisiologia e Bioquímca, pela amizade, pelos abraços e seus substitutos, pela transmissão de valores e conhecimentos tão importantes e pelas longas discussões - enzimáticas e afins! Prof. Jorge Marques da Silva, por todos os incentivos que transmitiu, directa ou indirectamente, pelos conhecimentos de stress hídrico e de fisiologia - alguns um tanto ou quanto fluorescentes! Dra. Ana Rita Matos pela lufada de ar fresco que trouxe ao CEB e pela capacidade de “apaziguação”. Ana Sofia Soares pelas aventuras e alegrias partilhadas no início de um percurso que acabou por tomar rumos diferentes! Prof. Cristina Cruz pela energia contagiante e pela recepção de braços abertos nas rápidas “visitas” ao laboratório da Ecologia! Prof. Cristina Máguas e Dr. Rodrigo Maia pelo apoio na determinação de composições isotópicas. Prof. Lia Ascensão pelo acolhimento no “cantinho da Microscopia” e pela transmissão de valores morais e científicos! Prof. Renata Meira (visitante do Brazil!) pela ajuda sempre pronta e pelo bom humor contagiante! Prof. Rui Malhó, Rui, pelo despertar de uma energia conquistadora e pela forma diferente de iniciar o dia, com sorrisos partilhados!! Manuela Lucas, Manelita, por ser uma segunda mãe, por tomar conta de mim, por todas as ajudas e por ser a maior mestra na “lei do desenrasque” – arranja sempre uma solução para o problema do próximo!
All the researchers and colleagues at Rothamsted Research! John Andralojc for all the jokes and the help with Rubisco-related measurements and Pippa Madgwick for having a nice and comforting word always ready! Jeroni Galmés for sharing the hot lab, for the “paella” and for the company in beautiful Scotland! Marcela Baúdo, for being another Speedy Gonzalez (!!!) and for the deep breaths shared while planning drought experiments... Guy Kiddle for the advices on the HPLC, on the amino acid identification and also the ones on scientific life! Till Pellny for the technical, scientific and life advices and, most of all, for having fun teasing me!!! Simon Driscoll for the technical support on the gas-exchange measurements and for the knowledge transmitted on the subject. Also for sharing his life story and his music! Riekert van Heerden for the advices on proline estimation, on lab working and energy level maintenance!!! Richard Parkinson, Kate Rydlewski and Steve Harvey, who helped me to take care of my “little girls” in the greenhouse 28, compartment 106!!! Stephen Powers for all the statistical analysis and advices, the high-horses and the long (but productive) discussions, the support and comforting words and, especially, for Believing me as a scientist from the very first moment!
Amigos que conheci na FCUL! Entre os quais… André, pela loucura constante, Márcia pela boa disposição e energia positiva e Elsita pelos mimos tão gostosos! Cátia pela partilha de suspiros durante frases escritas e outros rabiscos que tal! Céu pelo melhor sorriso da manhã na FCUL! All the good friends I met abroad! Namely… Rui for being my salvation at the Manor! Isabel for being a good listener and a good advisor, on PhDs and Life! Marta for sharing the experience on stress studies, Anneke for being a company in the late hours, Hiro for being pure, Francesca for being like a sister, Alexia for the joy! Maria Paz for being crazy, Nisreen for being a good friend, Ivânia and Luis for making part of the Portuguese mafia at RRes! Salvador for the sports company and for the friendship! Duncan for being such a good listener!!! My chocolate supplies will always be there… Eleonora for the smile and the NB copies! Tanya for the contagious energy!!!
Amigos em Casa... Sílvia Duarte, conselheira de saúde! Catarina Oliveira, assitente de imagem: a beleza é um conceito relativo! Bruno Serrão, assistente de informática – o que seria do meu portátil sem as tuas consultas terapêuticas!!! Ana Francisco, Anita, mesmo que distantes, não nos ausentamos de todo, estamos lá e sabemos disso! Foi um prazer enfrentar o microscópio contigo!!! Ana Catarino, Nocas, pela vontade contagiante de vencer nesta conquista por um lugar ao sol e de conhecer o mundo lá fora! Inês Bruno, por estar sempre a meu lado neste percurso que temos feito pela Vida - de alguma forma sei que vais estar sempre lá!
Friends at my second Home! Petra Bleeker, Peet, fofa, for being my older sister!!! Thanks for all the advices!!! Samuel Doufur, Sam, for all the Chemistry explanations, for the secret supplies… Also for the culture lessons and cooking sessions, for the constant company and support, even during the worst hours… and, most of all, for being such a nice friend!
Miguel pelos bons momentos que passámos juntos, o percurso de vida que caminhámos lado a lado, muitas vezes afastados... O apoio incontestável em todas as decisões difíceis, mesmo que muito discutidas e refutadas! A lição de vida que ficou e o carinho que permanece, sempre. À minha família agradeço sobretudo a compeensão. Não é fácil colocar em palavras o quão importante foi para mim o apoio constante que recebi da vossa parte e a força que me dá, todos os dias, o Amor que nos une! A Cat e a Ju, pataca e pataquinha, que são crianças a sério, percebem o quanto as amo e aceitam que vá “lá para a Inglaterra”! O Daniel partilha uma certa estória e tem crescido muito rápido nesta Vida! Mãe, unidas pela distância e partilhando aquela forma especial de ver e de sentir! Pai… o que sou hoje resulta em grande parte daquilo que me ensinaste a Ser!
“Wisdom, Happiness and Courage
Are not waiting somewhere (…) at the end of a straight line; They’re part of a continuous cycle that begins right here. They’re not only the ending, but the beginning as well. The more it snows, the more it goes on snowing.”
“A Sabedoria, a Felicidade e a Coragem
Não estão à espera algures (…) ao fundo de uma linha recta; São parte de um ciclo contínuo que começa aqui mesmo. Não são apenas o fim, mas também o princípio.
Quanto mais neva, mais continua a nevar.”
CONTENTS
Summary ... 1
Resumo ... 3
Chapter I. General introduction and objectives ... 7
C4 photosynthetic metabolism ... 17
Drought stress ... 23
Objectives of the Thesis ... 31
References ... 32
Chapter II. Water relations and leaf anatomy of C4 grasses ... 47
Abstract ... 49
Introduction ... 50
Material and methods ... 53
Results ... 59
Discussion ... 71
References ... 79
Chapter III. Photorespiration and C4 photosynthesis under drought stress ... 85
Abstract ... 87
Introduction ... 88
Material and methods ... 91
Results ... 98
Discussion ... 107
References ... 115
Chapter IV. C4 enzymes in drought-stressed grasses... 121
Abstract ... 123
Introduction ... 124
Material and methods ... 127
Results ... 131
Discussion ... 136
References ... 140
Chapter V. Rubisco from C4 grasses under drought stress .. ... 145
Abstract ... 147
Introduction ... 148
Material and methods ... 152
Results ... 159
Discussion ... 167
References ... 172
Chapter VI. General discussion and conclusions ... 179
Concluding remarks ... 192
Future perspectives ... 193
LIST OF SYMBOLS AND ABBREVIATIONS USED
A net CO2 assimilation rate
Ac net CO2 assimilation rate calculated from quadratic expression for enzyme-limited
photosynthesis
An net CO2 assimilation rate calculated from the asymptotic exponential curve applied to the
variation of A with Ci
ABA abscisic acid
ACC 1-aminocyclopropane-1- carboxylic acid ADP adenosine 5’-diphosphate
Ala alanine
AlaAT alanine aminotransferase AMP adenosine monophosphate
Asn asparagine
Asp aspartate
AspAT aspartate aminotransferase ATP adenosine 5’-triphosphate Bicine N,N-bis(2-hydroxy-ethyl)glycine
BS bundle sheath
Ca atmospheric CO2 concentration (in the gas phase) Ci CO2 concentration in the intercellular air-spaces Cm CO2 concentration in the mesophyll cells Cs CO2 concentration in the bundle sheath cells
CA carbonic anhydrase CA1P 2-carboxyarabinitol-1-phosphate CABP 2-carboxyarabinitol-1,5-bisphosphate Chl chlorophyll δ isotope composition DTT 1,4-dithiothreitol DW dry weight
EDTA ethylenediaminetetraacetic acid E4P erythrose-4-phosphate
γ* half the reciprocal of Rubisco specificity
gbs bundle sheath conductance to CO2 gi mesophyll conductance to CO2
gswa stomatal conductance to water vapour
Gln glutamine
Glu glutamate
Gly glycine
Hepes 4-(2-hydroxy-ethyl)-1-piperazine-ethanesulfonic acid HNV 5-hydroxy-L-norvaline (or 2-amino-5-hydroxypentanoic acid)
FW fresh weight
GOGAT ferredoxin (Fd-) or NADH-glutamate synthase
Ko Michaellis-Menten constant of Rubisco for O2 Kp Michaellis-Menten constant of PEPC for CO2 KRuBP Michaellis-Menten constant of Rubisco for RuBP
LN2 liquid nitrogen
LSD least significant difference LWP leaf water potential M mesophyll
MDH malate dehydrogenase MEA monoethanolamine
Met methionine
MS moderate drought stress
NAD-ME NAD-malic enzyme
NAD+ nicotinamide-adenine dinucleotide (oxidized) NADH nicotinamide-adenine dinucleotide (reduced) NADP-ME NADP- malic enzyme
NADP+ nicotinamide-adenine dinucleotide phosphate (oxidized)
NADPH nicotinamide-adenine dinucleotide phosphate (reduced) n.d. not determined
O O2 partial pressure in the bundle sheath and mesophyll cells
O2 oxygen
OAA oxaloacetate
P probability or level of significance PDBP D-glycero-2,3-diulose-1,5-bisphosphate PEG polyethylene glycol
PEP phosphoenolpyruvate
PEPC phosphoenolpyruvate carboxylase PEPCK phosphoenolpyruvate carboxykinase
PG 2-phosphoglycolate
PGA 3-phosphoglycerate
Phe phenylalanine
Pi orthophosphate
PPdK pyruvate,orthophosphate dikinase PPFD photosynthetic photon flux density
PPi pyrophosphate
Pr rate of photorespiration PVP polyvinylpyrrolidone
Rd leaf mitochondrial respiration
Rm mesophyll mitochondrial respiration
R2 percentage of variance accounted for by a model
REML Residual Maximum Likelihood ROS reactive oxygen species
Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase Ru5P ribulose-5-phosphate
RuBP ribulose-1,5-bisphosphate RWC leaf relative water content
S non-watered or drought-stressed s2 residual mean square
s.e. standard error of the mean SED standard error of differences
Ser serine
SLA specific leaf area
SS severe drought stress SWC soil water content
TCA tricarboxylic acid TFA trifluoroacetic acid
TW turgid weight
Vc rate of Rubisco carboxylation
Vcmax maximum Rubisco carboxylation activity Vmax maximal activity
Vo rate of Rubisco oxygenation
Vomax maximum Rubisco oxygenation activity Vp rate of PEPC carboxylation
Vpmax maximum PEPC carboxylation activity
Vphysiol physiological activity
WUE water use efficiency WWP water weight in pot
SUMMARY
Drought stress is one of the major constraints to plant photosynthetic performance. With increasing water scarcity in many areas of the world the understanding of fundamental drought-stress physiology and biochemistry is crucial to optimize the exploitation of plant responses and improve water use efficiency. The CO2-concentrating mechanism present in C4 plants limits
photorespiration and increases their photosynthetic performance. The physiological and biochemical responses to gradually-induced water deficit of three C4 grasses with different
decarboxylating mechanisms were compared. Paspalum dilatatum Poir. (NADP-ME) was less resistant to drought than Cynodon dactylon Pers. (NAD-ME) and Zoysia japonica Steudel (PEPCK). The faster leaf dehydration in the first species reflects the higher water requirement associated with its high productivity. Leaf structure of all three species, especially C. dactylon and Z. japonica, showed advantageous characteristics to cope with xeric environments. In all three species photosynthesis and stomatal conductance decreased under water deficit but showed continued CO2 assimilation even when leaf dehydration was severe. Photorespiration, as
evaluated by CO2 exchange rates at different concentrations of CO2 and O2, by mechanistic
modelling of C4 photosynthesis and by changes in amino acids in a 30 s dark period, remained
slow under drought conditions, supporting the photosynthetic efficiency of the CO2
-concentrating mechanism. Moderate drought stress did not affect dramatically the carboxylating and decarboxylating enzyme activities but caused changes in the regulation of PEPC and Rubisco. Considerable activity of PEPCK was found in all three species suggesting its involvement in the C4 photosynthetic pathway, acting as a secondary decarboxylase in P. dilatatum and C. dactylon, or in other non-photosynthetic processes occurring in the leaves of the three C4 grasses. An unusual hydroxylated amino acid was drought-inducible and its
potential benefits for drought resistance should be further investigated.
KEYWORDS:
Water deficit, C4 photosynthesis, Photorespiration, Paspalum dilatatum, Cynodon dactylon, Zoysia japonica
RESUMO
O défice hídrico é um dos factores que mais limitam a produtividade vegetal. A escassez de água é um problema que atinge proporções cada vez maiores em muitas áreas do planeta e tende a ser exacerbado devido às reconhecidas alterações climáticas. A implementação de práticas de rega mais correctas, envolvendo monitorização das condições hídricas do solo e/ou das plantas, bem como a utilização de espécies e variedades de plantas melhor adaptadas a condições de baixa disponibilidade de água poderão contribuir para uma mais eficiente gestão dos recursos hídricos. O conhecimento da fisiologia e bioquímica em condições de stress são fundamentais para que as respostas adaptativas das plantas a condições xéricas possam ser exploradas e a eficiência do uso da água melhorada.
A pressão exercida sobre os diferentes utilizadores de água é assim cada vez maior e afecta diversas áreas, incluindo a agricultura e o turismo. O crescente número de campos de golfe em Portugal implica gastos hídricos adicionais que são muitas vezes criticados, sobretudo nos verões em que a seca é mais severa e a necessidade de água para a produção agrícola torna o desporto uma necessidade supérflua. A utilização de espécies e variedades de relvas mais adequadas (que gastem menos água mantendo elevada performance) aliada a práticas de rega que envolvam, por exemplo, reutilização de águas residuais irá contribuir para uma maior eficiência do uso da água disponível.
O mecanismo de concentração de CO2 presente nas plantas com metabolismo
fotossintético em C4 torna-as menos sensíveis ao oxigénio e a resultante limitação da
fotorrespiração nestas plantas contribui para o aumento da eficiência do uso de água. As relvas C4 estão, por esse motivo, geralmente associadas a uma maior resistência ao défice hídrico, mas
não há necessariamente uma relação directa entre eficiência de uso de água e resistência à seca. A distribuição das espécies C3 e C4 sugere que a temperatura é o principal factor que se
correlaciona com a ocorrência de cada uma das variantes fotossintéticas. A precipitação, por outro lado, parece estar relacionada coma distribuição diferencial dos vários subtipos de fotossíntese em C4. As espécies do subtipo NADP-ME (enzima málico dependente do NADP)
são geralmente mais abundantes em áreas com maior precipitação e as espécies do subtipo NAD-ME (enzima málico dependente do NAD) estão mais representadas em zonas mais secas, enquanto que a distribuição das espécies do subtipo PEPCK (fosfoenolpiruvato carboxicinase) em relação aos gradientes de precipitação apresenta um padrão mais incerto.
As respostas fisiológicas e bioquímicas ao défice hídrico foram estudadas em três espécies de relvas C4 dos diferentes subtipos metabólicos: Paspalum dilatatum Poir.
(NADP-ME), Cynodon dactylon Pers. (NAD-ME) e Zoysia japonica Steudel (PEPCK). As três espécies foram comparadas em termos da presença de mecanismos de resistência ao stress envolvendo a manutenção dos conteúdos hídricos da planta ou a tolerância a condições de desidratação foliar. Uma abordagem inicial envolveu o estudo das relações hídricas e da estrutura foliar, a que se seguiu o estudo das trocas gasosas, da fotorrespiração e de outros aspectos do metabolismo fotossintético relativos aos processos de carboxilação e descarboxilação.
As plantas de P. dilatatum foram menos resistentes ao défice hídrico, observando-se uma diminuição mais rápida do teor hídrico relativo das suas folhas que nas plantas de C. dactylon e Z. japonica. As folhas das três espécies são caracterizadas por uma estrutura foliar especializada, com anatomia foliar Kranz, que permite uma maior eficiência da utilização dos recursos hídricos. A espécie Z. japonica, em particular, tem paredes celulares mais rígidas e possui maior quantidade de esclerênquima, o que se traduz numa maior rigidez e dureza das folhas. A rigidez celular permite a diminuição da pressão de turgescência quando o teor hídrico começa a diminuir, promovendo o abaixamento do potencial hídrico das folhas, que foi mais acentuado em Z. japonica e pode ser visto como uma estratégia que permite o aumento da tomada de água do solo à medida que a sua disponibilidade diminui. A capacidade de acumulação de osmólitos, incluindo a prolina e outros aminoácidos, permite aumentar a pressão osmótica e contribuem assim para a diminuição do potencial hídrico. No entanto, o aumento de aminoácidos como a prolina, metionina, valina, fenilalanina, isoleucina e leucina sugere também o seu envolvimento directa ou indirectamente (através da produção de compostos do metabolismo secundário) em mecanismos de defesa, nomeadamente protecção contra espécies reactivas de oxigénio e utilização de poder redutor em excesso. Nas folhas de C. dactylon e Z. japonica foi identificado um aminoácido hidroxilado não-proteico que não tinha sido previamente descrito como estando presente em folhas e que apresenta características que devem ser exploradas. O aminoácido 5-hidroxi-L-norvalina (HNV) ocorre em folhas de plantas de Z. japonica bem irrigadas e o seu conteúdo aumenta com o défice hídrico. Em C. dactylon HNV não está presente nas folhas de plantas bem irrigadas mas surge e o seu conteúdo aumenta acentuadamente com a desidratação foliar. Em P. dilatatum o aminoácido não está presente e não é induzido pelo stress. Associando este comportamento à elevada resistência à seca de C. dactylon e Z. japonica sugere-se que este aminoácido poderá estar intimamente associado ao mecanismo de defesa destas plantas.
A anatomia foliar das plantas das três gramíneas não foi muito alterada em condições de stress e sugere que as folhas destas espécies apresentam características intrínsecas que lhes permitem uma melhor adaptação a condições de reduzida disponibilidade de água, conferindo-lhes uma maior eficiência no uso de água. O aumento da proporção de células do mesófilo em
relação às células da bainha perivascular em P. dilatatum e Z. japonica em condições de stress sugere alguma capacidade de adaptação ao défice hídrico, mas a ausência de maiores alterações morfológicas induzidas pelo stress reflecte a baixa plasticidade fenotípica das três espécies.
O mecanismo de concentração de CO2 característico do metabolismo fotossintético em
C4 limita a reacção oxigenativa da Rubisco e, consequentemente, a taxa de fotorrespiração. No
entanto, o fecho dos estomas em condições de défice hídrico pode levar à diminuição da concentração intercelular de CO2 e ao aumento da fotorrespiração. Nas três gramíneas estudadas
houve uma diminuição da taxa de fotossíntese e da condutância estomática com a desidratação foliar, mas as trocas gasosas continuaram mesmo quando o teor hídrico diminuiu para valores inferiores a 80%, correspondente a cerca de 10 dias sem irrigação e um reduzido conteúdo de água no solo. O decréscimo da fotossíntese com o aumento da concentração de oxigénio e a diminuição do conteúdo em glicina ao fim de um período de 30 s em escuridão evidenciaram a presença de taxas de fotorrespiração, embora muito mais lentas nestas três espécies C4 que em
plantas C3. A análise conjunta das trocas gasosas de CO2 a diferentes concentrações de CO2 e O2,
incluindo o ponto de compensação para o CO2 e sensibilidade da fotossíntese ao oxigénio e a
aplicação de um modelo de fotossíntese em C4 aos dados experimentais revelaram a manutenção
de taxas de fotorrespiração lentas em condições de défice hídrico. A variação do conteúdo em aminoácidos, especialmente glicina, na fracção solúvel das folhas, suportou a capacidade das três espécies estudadas para limitar a velocidade de oxigenação da RuBP mesmo em condições de défice hídrico. O aumento do conteúdo em glicina, serina e etanolamina com a desidratação foliar bem como os resultados comparativos da modelação das trocas gasosas em condições controlo e de stress forneceram os únicos indícios para um ligeiro aumento do metabolismo fotorrespiratório em relação à fotossíntese.
Para uma maior eficiência do metabolismo fotossintético em C4 é necessário haver uma
boa coordenação entre a carboxilação primária do CO2, levada a cabo pela PEPC no mesófilo, e
a subsequente descarboxilação dos ácidos C4 na bainha perivascular. As actividades da PEPC,
NADP-ME, NAD-ME e PEPCK não foram muito afectadas pelo défice hídrico e consideradas no seu conjunto sugeriram uma maior eficiência fotossintética em C. dactylon que nas outras duas espécies. O estado de fosforilação da PEPC aumentou, revelando uma potencial resposta adaptativa das três gramíneas C4 ao stress no sentido de tornar a carboxilação primária do CO2
mais eficiente em condições que promovem o fecho dos estomas, reduzindo a disponibilidade de CO2 a nível intercelular. Curiosamente, observaram-se consideráveis actividades da PEPCK nas
três espécies, pertencentes a cada um dos subtipos metabólicos de plantas C4, revelando que a
(subtipos NADP-ME e NAD-ME, respectivamente) ou desempenhar funções no metabolismo não-fotossintético das três espécies. As potencialidades associadas à presença de elevadas actividades da PEPCK em gramíneas C4 dos vários subtipos metabólicos devem ser investigadas
em maior detalhe no sentido de aumentar a compreensão do metabolismo fotossintético em C4.
Em condições de défice hídrico, a diminuição das taxas de assimilação de CO2 implica
que a actividade da Rubisco seja regulada de modo a manter o equilíbrio entre os diferentes processos do metabolismo fotossintético. A diminuição do conteúdo em RuBP nas folhas desidratadas das três espécies sugere que a regeneração deste substrato é afectada em condições de défice hídrico, possivelmente devido a danos ao nível do ciclo de Calvin ou reduzida síntese de ATP, limitando a assimilação de CO2. O aumento de inibidores nas mesmas folhas sugere que
estes se ligam à Rubisco em condições de stress protegendo a enzima contra o dano proteolítico, não se observando alterações no conteúdo em Rubisco.
A aplicação de modelos de fotossíntese para simular os efeitos induzidos por alterações ambientais tem sido comprometida pela falta de conhecimento de vários parâmetros fundamentais e pela quantidade de pressupostos inerentes á sua utilização. As constantes cinéticas para as actividades carboxilativa e oxigenativa da Rubisco parcialmente purificada de cada uma das três gramíneas C4 estudadas irá permitir aumentar o rigor e a precisão com que se
fazem simulações baseadas nestes modelos. Em comparação com a espécie C3 utilizada como
modelo, o trigo, a Rubisco de P. dilatatum, C. dactylon e Z. japonica é caracterizada por factores de especificidade mais baixos, e constantes de Michaelis-Menten para o CO2 e o O2 e
velocidades de carboxilação máxima mais elevadas.
As três espécies de gramíneas C4 estudadas apresentam elevada eficiência fotossintética,
sobretudo C. dactylon, e elevada resistência ao défice hídrico, sobretudo C. dactylon e Z. japonica. O metabolismo fotossintético é regulado de modo adaptativo e a limitação da taxa de fotorrespiração mantém-se em condições de stress. As potencialidades associadas ao aumento do conteúdo em HNV com a desidratação foliar e à presença de elevadas actividades da PEPCK nas três espécies pertencentes a diferentes subtipos metabólicos devem ser exploradas. As constantes cinéticas da Rubisco determinadas para gramíneas C4 permitirão o rigor na simulação das
respostas do metabolismo fotossintético a diversas condições ambientais.
PALAVRAS-CHAVE:
Défice hídrico, Fotossíntese C4, Fotorrespiração Paspalum dilatatum, Cynodon dactylon, Zoysia japonica
Chapter I.
GENERAL INTRODUCTION AND OBJECTIVES
Climate change and water shortage – the problem!
Water is one of the most important factors limiting plant growth, development and survival. In particular in Mediterranean climates, which are characterized by long hot and dry periods, water availability is the major limitation to plant productivity (Turner 2004). The decrease of fresh water availability is one of the most serious environmental problems of the planet, as recognized by the United Nations Environment Programme (Global Environment Outlook: Environment for Development, Geo-4 report 2007). The number of dry days per year is expected to raise in many areas of the globe (Petit et al. 1999), exacerbating the problem.
The efficient management of water resources by agricultural and recreational systems is therefore an outstanding priority in many regions of the world and there is increasing pressure on the different irrigators to increase the efficiency of water use by crops and pastures or lawns. Golf courses are probably the most criticized water-users, especially during summer-droughts, in the USA and in some European countries, including Portugal. This is mostly because the game is not considered a ‘primary need’. Even considering the enormous importance in terms of tourism and development, serious concerns arise in terms of water consumption. The tourism sector has been recognised as one of the key water users in Europe where there is big potential for water savings, including the use of more efficient irrigation techniques and rain water harvesting in golf courses (MAOTDR 2007b). In America great investments have been made in order to promote increased water use efficiency at the level of the golf industry (Snow 2001), namely through the use of varieties of turfgrass that require less water, better irrigation techniques and management practices (e.g. monitoring the soil moisture for scheduling irrigation), and the use of alternative water sources.
Deficit irrigation techniques have been successfully applied in the field to improve water use efficiency by some crops and might have further application in water-limited environments (Costa et al. 2007; Fereres & Soriano 2007). For instance, the strategy of partial rootzone drying, which involves simultaneous exposition of the roots to wet and drying soil, allows the decrease of stomatal conductance, decreasing water loss, without affecting plant performance (Davies et al. 2002; Santos et al. 2003). The use of secondary, non-potable waters for the irrigation of turfgrasses has been suggested as another possible alternative (Snow 2001; Marcum 2006) and water re-utilization is already implemented in many golf courses in the South of Portugal
(MAOTDR 2007a). Irrigation systems controlled by soil moisture sensors can also be used to minimize water waste by matching turfgrass requirements (Pathan et al. 2007).
The use of plant species and cultivars better suited to the environment can also improve water use efficiency and plant production and yields in water-limited regions (Turner 2004). As recently proposed by Marcum (2006), turfgrasses with high salinity tolerance allow the use of saline, non-potable waters for irrigation. Snow (2001) reported the use of stress-tolerant bermudagrass (Cynoodn dactylon) cultivars with low water requirements yielding considerable water savings in American golf courses, and also the successful irrigation of the extremely salt-tolerant and highly productive seashore paspalum (Paspalum vaginatum) with salted waters. It is also important to note that, in what concerns the golf courses in particular, there are different areas associated with different game functions and the turfgrasses used in each of these should correspond to the functional needs associated (Bernardes da Silva et al. 2008).
The grass family and the C4 photosynthetic pathway
The multifaceted contribution of the Poaceae to the world economy is the base for the superlative importance of this plant family. The grass family includes the cereals (such as wheat, rice, maize and sorghum), the most important source of sucrose in the world (sugarcane), and the forage and turf grasses, which are the backbone of sustainable agriculture (Jones 1985). The paramount importance of the forage and turf grasses arises from the economic benefits associated with its use in golf courses and other recreational areas, the sustainability of live stocks and wild animals, and from their contribution to the soil conservation and environment protection.
Grasses are commonly classified as cool-season and warm-season, reflecting low (ca. 15º-25ºC) or high (ca. 25º-35ºC) optimal daytime growth temperatures, respectively. The distinct physiological characteristics between the two types of grasses result from the presence of the C4
photosynthetic pathway that elevates the CO2 concentration at the site of carboxylation in the
warm-season (C4) grasses (Furbank & Hatch 1987). C4 photosynthesis occurs in nearly half of
the species in the Poaceae (Hattersley 1988; Sage et al. 1999a) and some of the world’s most important crop and turf species are C4 grasses (see Jones 1985; and Brown 1999). Warm-season
C4 grasses, by opposition to cool-season C3 species, are characterized by performing better at
high irradiance levels and high temperatures (Johnston 1996), showing higher rates of photosynthesis than their C3 counterparts in full sunlight and at temperatures above 30ºC (Brown
1999). C4 grasses have generally higher water use efficiency (WUE) due to an efficient
comparison by Gherbin et al. (2007) revealed that warm-season C4 grasses produce higher yields
than cold-season C3 grasses in warm dry summer conditions like those observed in the
Mediterranean climate and in some regions of the USA and Australia.
The distribution of C3 and C4 grasses in several regions of the world suggests that
temperature is the climatic variable best correlated with the relative occurrence of grass species of the two photosynthetic pathways (Henderson et al. 1995; Sage et al. 1999b). As shown by Cabido et al. (1997), C4 species tend to dominate in areas characterised by warmer temperatures
and their distribution is therefore dependent on a latitudinal and altitudinal gradient. Sage et al. (1999b) noted that aridity is not a prerequisite for C4 dominance over C3 and the success of C4
species depends essentially on the presence of warm temperatures and high light intensities, resulting in a high representation of this photosynthetic pathway in tropical and subtropical regions, where more than two thirds of all grasses are C4.
The presence of a CO2-concentrating mechanism makes C4 photosynthesis more
competitive in conditions that promote photorespiration, like high temperatures and low intercellular CO2 concentrations. Consequently, it is generally assumed that C4 species will
perform better in warm habitats and conditions that promote stomatal closure, including decreased water availability. Importantly, Sage et al. (1999b) noted that the seasonality of precipitation plays an important role and its occurrence during the warm season tends to favour C4 dominance. Nevertheless, several reports suggest that C4 grasses may dominate in areas
where precipitation decreases in the warmer summer months (see Cabido et al. 2008) and their exploitation can therefore bring advantage in terms of water savings.
The diversification of the anatomical-biochemical variants of C4 photosynthesis might be
related with natural selection pressures of changes in rainfall (Hattersley & Watson 1992), as precipitation gradients seem to be the major determinant for the relative distribution of the three classical variants of C4 photosynthesis. These are named after the main decarboxylating enzyme
in each pathway (Gutierrez et al. 1974; Hatch et al. 1975): NADP-malic enzyme (NADP-ME), NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). The bio-geographical distribution of C4 species with different decarboxylation mechanisms in several
regions of the world (e.g. Hattersley 1992; Cabido et al. 2008) shows that NADP-ME species are relatively more abundant in areas with higher annual rainfall whereas NAD-ME species predominate in arid zones, and PEPCK seem to have a less clear pattern of association with precipitation gradients. Brown (1999) refers that most of the cultivated C4 species with
agronomic importance are NADP-ME, possibly as a consequence of their occurrence in wetter areas, the first to be exploited during colonization. In the current climate conditions and
assuming the future perspectives, the implementation of some potentially more drought-resistant NAD-ME species might bring considerable advantage in terms of better use of water resources. The positive and negative correlation of NAD-ME and NADP-ME species with aridity, respectively, may be related with enhanced water use efficiency in the former species (Ghannoum et al. 2002). However, it is unclear if this is due to the functional differences between the two variants of C4 photosynthesis. Most grasses from the subfamily Chloridoideae
are C4 and belong to the subtypes NAD-ME or PEPCK, whereas the subfamily Panicoideae is
photosynthetically more variable, with representatives of C3, intermediate C3-C4 and C4
photosynthesis (Hattersley & Watson 1992). All three biochemical subtypes of C4 grasses occur
in the Panicoideae, but the great majority of C4 species in this subfamily are NADP-ME (Taub
2000). The distribution of the subfamilies Panicoideae and Chloridoideae is also strongly correlated with the precipitation gradients (Taub 2000; Cabido et al. 2008), suggesting that characteristics other than the biochemistry of photosynthesis may be responsible for the geographical patterns observed, possibly reflecting some divergent patterns associated with the multiple origins of C4 grasses (Kellogg 1999).
In addition to the specialised photosynthetic biochemistry, the leaves of most C4 grasses
show anatomical modifications associated with the functionality of the CO2-concentrating
mechanism. These characteristics are referred to as Kranz anatomy (see review by Dengler & Nelson 1999). The term ‘Kranz’ refers to a wreath of cells surrounding the vascular tissues and was first used by Haberlandt (1882) who initially referred to the presence of distinct leaf anatomies and recognized that grasses could be divided into two groups and these were related with their ecological adaptation. A suite of subtype-specific anatomical characteristics has been further associated with each of the decarboxylation mechanisms (Prendergast & Hattersley 1987; Dengler et al. 1994). Most C4 grasses belong to the ‘classical’ biochemical-anatomical subtypes
(Hattersley & Watson 1992), but some variations occur in nature (e.g. Prendergast et al. 1987). As suggested by Hattersley (1992), differences in leaf structure could possibly be associated with differential ability of different grass species to cope with decreased water availability.
C4 species and climate change
Although C4 plants represent less than 4% of the terrestrial plant species (Sage et al. 1999a) they
contribute about one-quarter of the primary productivity of the planet and a large fraction of the primary production consumed by humans, directly or not, is derived from C4 crops and pastures
(Brown 1999). The specialized leaf structure, with a high density of vascular bundles, makes C4
(Wilson & Hattersley 1989; Scheirs et al. 2001), but there is no consistent selection against C4
and in some areas C4 grasses are actually preferred in comparison to C3 (see Heckathorn et al.
1999). The increased interest in C4 grass pastures comes from the fact that they are more
productive and may be more drought resistant (Blaikie et al. 1988). The same reasons provide evidence for their preferential use as turf in recreational areas. Additionally, the potential use of C4 grasses as biofuels in energy production systems was recently outlined (Samson et al. 2005).
Climate change has remarkable consequences on biodiversity, species’ distribution and their relative abundance. Water availability, in particular, is one of the most relevant environmental factors affecting plant survival, productivity and distribution. The effects of increasing atmospheric CO2 on growth and photosynthesis in C3 and C4 plants are still
controversial and associated with a great level of uncertainty (Chen et al. 1996; Campbell & Smith 2000; Ainsworth & Long 2005; Korner 2006; Long et al. 2006; Soares et al. 2008), but the predicted temperature rise is likely to favour C4 photosynthetic performance and
competitiveness and result in increased dominance of grasses with this photosynthetic pathway (Henderson et al. 1995; Sage & Kubien 2003). Changes in rainfall scenarios are not as easy to predict as those of temperature and involve more uncertainty but are likely to affect the relative distribution of C4 grass species, with increased NAD-ME proportional occurrence in drier areas.
The comprehensive understanding of the C4 photosynthetic metabolism and the response
of plants with the C4 pathway to the environment are crucial if the best advantage is to be taken
from their potentialities. Recent progress has been made on the understanding of C4
photosynthesis in dicotyledon species e.g. (Voznesenskaya et al. 2007) and suggestion has been made to adopt a species from the genus Cleome as a C4 model system (Brown et al. 2005). None
the less, there is great advantage on the use of C4 grass species. It is difficult to outcome the
economic, agronomic and ecological importance of the Poaceae. The C4 photosynthetic pathway
was first discovered in the grass family and photosynthetic variation within the Poaceae is comprehensively understood (see review by Hattersley & Watson 1992). Most C4 grasses have
been biochemically typed in terms of photosynthetic variant and a checklist has been provided by Hattersley some twenty years ago (Hattersley 1988). Therefore, grasses provide the ideal system for the study of C4 photosynthesis under changing environments.
The C4 grasses Paspalum dilatatum, Cynodon dactylon and Zoysia japonica
The three warm-season C4 grasses studied in the present work (Figure I.1) have been previously
classified as belonging to each of the different biochemical subtypes of C4 photosynthesis. Paspalum dilatatum Poir. is a NADP-ME species (Usuda et al. 1984), Cynodon dactylon Pers. is a NAD-ME species (Hatch & Kagawa 1974) and Zoysia japonica Steudel is a PEPCK species (Gutierrez et al. 1974). P. dilatatum belongs to the subfamily Panicoideae, while C. dactylon and Z. japonica belong to the subfamily Chloridoideae (Watson & Dallwitz 1992). The genus Paspalum originated in South America, Cynodon in Africa and Zoysia in Southeastern Asia (Brown 1999).
Paspalum dilatatum Cynodon dactylon Zoysia japonica
Paspalum dilatatum Cynodon dactylon Zoysia japonica
Figure I.1. Plants of the three C4 grass species in the greenhouse during the early stages of development.
Photograph was taken ca. two (P. dilatatum and C. dactylon) or four (Z. japonica) weeks after sowing.
Dallisgrass (P. dilatatum), bermudagrass (C. dactylon) and zoysiagrass (Z. japonica) are warm-season perennial species used for turfgrass purposes throughout the world (Brown 1999). Additionally, the first two species are important forage and cultivated pasture grasses and C. dactylon is also one of the world’s most serious weeds (Jones 1985; Brown 1999).
The species P. dilatatum, native from South America, is an important forage grass in the subtropical and warm regions of the world, mainly due to its high nutritive value (Venuto et al. 2003). Since long ago, dallisgrass has been a dominant pasture in Australia, especially during the summer season (Stockdale 1983). Its great value is also derived from its cold tolerance and ability to survive frosts in winter (Rowley 1976). Andrews & Crofts (1979a) evaluated the possibility of replacing pastures of dallisgrass by bermudagrass in order to increase the growing
season recognizing the value of the latter in areas with low frost incidence. However, both the highly productive, wide-temperature- and grazing-tolerant dallisgrass as well as the promising bermudagrass were outyelded by an improved cultivar of Pennisetum clandestinum (Pearson et al. 1985). None the less, dallisgrass is still an important pasture forage and progress has recently been observed in the improvement of its forage yields (Venuto et al. 2007). In some modern prairies, dallisgrass can become a weed to bermudagrass turf (see Henry et al. 2007).
The outstanding economic importance of C. dactylon results from the wide distribution throughout tropical and subtropical areas and the enormous variability of the species (Taliaferro 1995). The recognition of ecotypes with different characteristics of establishment and persistence (Andrews & Crofts 1979a) and of digestibility (Andrews & Crofts 1979b), suggested these could be used most favourably to different purposes (e.g. control of soil erosion, feedstock, etc). Bermudagrass, native from Africa, is the most widely used turfgrass (in lawns, golf courses and other sports fields) in tropical and subtropical regions of the world (Brosnan & Deputy 2008) and is used as forage for livestock (Starks et al. 2006). Additionally, C. dactylon can also be applied in the stabilisation of soils (Vignolio et al. 2002; Moreno-Espindola et al. 2007) and has been recently suggested as a potential energy crop for biofuel production (Boateng et al. 2007). However, C. dactylon is sensitive to shading and requires full sun for best performance (Guglielmini & Satorre 2002; Tegg & Lane 2004; Brosnan & Deputy 2008). The species is also cold-sensitive, but different lines are now being developed with improved freeze tolerance (Anderson et al. 2007). Bermudagrass is a target for genetic engineering for turf quality improvement (Li et al. 2005; Wang & Ge 2005).
The species Z. japonica, native from Japan, is sometimes called Japanese or Korean lawn grass (Duble 2002). Despite its slow growth rate, zoysiagrasses are shade tolerant and perform well as lawns, being widely used in golf courses and other sports fields (Deputy & Hensley 1999). Zoysia japonica is widely used in Japan and other countries of Asia as a turfgrass for sports fields and as a forage grass (see Cai et al. 2005 for references in Japanese!). Slow establishment of lawns is one of the major barriers for the use of zoysiagrass, but it was recently shown that different genotypes may establish faster (Patton et al. 2007). This grass species requires watering during lawn establishment but afterwards it is one of the most drought- and heat-resistant warm-season grasses (Deputy & Hensley 1999). Some contradictory references for its cold resistance are related with differences in freeze tolerance among genotypes of zoysiagrass (Patton & Reicher 2007) and the winter hardiness and high temperature tolerance suggest that zoysiagrass can adapt to a wide range of environmental changes (see White et al. 2001). Zoysiagrass is characterized by very stiff leaf blades due to high content in silica (Duble
2002) and is nearly as salt tolerant as bermudagrass due to the presence of salt secretion glands in their leaves (Marcum 1999). Additionally, zoysiagrass seems to be less sensitive to low nutrient supply than bermudagrass (Menzel & Broomhall 2006), with associated potential savings in fertilizers. Its recognized turf value makes zoysiagrass subject of genetic transformation (e.g. Ge et al. 2006).
The remarkable characteristics of P. dilatatum, C. dactylon and Z. japonica provide evidence for the potentialities associated with the understanding of the responses of these grass species to the environment, particularly to conditions of decreased water availability, and the functional significance of their C4 photosynthetic pathways under these conditions.
C4 PHOTOSYNTHETIC METABOLISM
Biochemistry and anatomy of C4 photosynthesis
The C4 photosynthetic pathway, with specialised biochemical and anatomical characteristics,
results in elevated CO2 concentrations at the site of the carboxylating enzyme,
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39), thereby increasing photosynthetic efficiency in conditions promoting high rates of photorespiration. The main features associated with C4 photosynthesis were identified in the two decades that followed the discovery of the C4
dicarboxylic acid pathway in the mid-1960’s (see historical overview by Hatch 1999).
In C4 grass leaves with Kranz anatomy, primary fixation of inorganic carbon by
phosphoenolpyruvate (PEP) carboxylase (PEPC; EC 4.1.1.31) occurs in the mesophyll (M) cells. The C4 acids formed are then transported to the bundle sheath (BS) cells, where they undergo
decarboxylation and the released CO2 is subsequently assimilated by Rubisco in the C3 pathway
(Hatch 1987; Kanai & Edwards 1999). PEPC is confined to the cytosol of M cells and Rubisco is confined to the chloroplasts of BS cells (Edwards et al. 2001). The specialized leaf structure (see Dengler & Nelson 1999), namely the chemical modification and increased thickness of the BS cell walls and the reduction of the exposure of BS surface area to intercellular spaces, decrease leakage of CO2 back to M cells so that CO2 accumulates (Furbank et al. 1989; Brown & Byrd
1993; Evans & von Caemmerer 1996; Jenkins 1997; Kiirats et al. 2002). C4 photosynthesis
saturates at lower CO2 concentrations than in C3 plants, essentially because the affinity of PEPC
for HCO3- is much higher than the affinity of Rubisco for CO2 (Kanai & Edwards 1999). The use
of C4 photosynthesis mutants provided evidence that the flux through this pathway is controlled
by several enzymes, with special emphasis on Rubisco, PEPC pyruvate Pi dikinase (PPdK, responsible for the regeneration of PEP in the M cells) (see review by Lea et al. 1999).
Three biochemical subtypes of the C4 photosynthetic pathway have been classically
defined according to the main enzymes responsible for the decarboxylation step (Gutierrez et al. 1974; Hatch et al. 1975): NADP-malic enzyme (NADP-ME, EC 1.1.1.40), NAD-malic enzyme (NAD-ME, EC 1.1.1.39) and PEP carboxykinase (PEPCK, EC 4.1.1.49). The three mechanisms of C4 photosynthesis were first described by Hatch (1987) and are summarised in Figure I.2.
These subtypes are distinguished by several aspects of leaf biochemistry and anatomy (see reviews by Dengler & Nelson 1999; Kanai & Edwards 1999), which are discussed in further detail in Chapter IV. The regulation of the C4 pathway, including the different subtypes, was
CO2 CO2 NADP+ NADPH Pyruvate CHLOROPLAST PEP HCO3
-OAA OAA Malate
NADP+ NADPH CHLOROPLAST PEP ATP + Pi AMP + PPi Pyruvate Malate Calvin Cycle
MESOPHYLL CELL BUNDLE SHEATH CELL
RuBP MDH PPdK Rubisco A NADP-ME Pi NADP-ME PEPC CA CO2 CO2 NADP+ NADPH Pyruvate CHLOROPLAST PEP HCO3
-OAA OAA Malate
NADP+ NADPH CHLOROPLAST PEP ATP + Pi AMP + PPi Pyruvate Malate Calvin Cycle
MESOPHYLL CELL BUNDLE SHEATH CELL
RuBP MDH PPdK Rubisco A NADP-ME Pi NADP-ME PEPC CA NAD+ OAA Calvin Cycle CO2 CO2 Pyruvate CHLOROPLAST PEP HCO3 -OAA CHLOROPLAST PEP ATP + Pi AMP + PPi
MESOPHYLL CELL BUNDLE SHEATH CELL
RuBP PPdK Rubisco B NAD-ME Pi Malate Aspartate Aspartate Alanine NADH Pyruvate Pyruvate Pyruvate Alanine (NH2) MDH NAD-ME MITOCHONDRION AspAT AlaAT (NH2) AspAT AlaAT PEPC CA NAD+ OAA Calvin Cycle CO2 CO2 Pyruvate CHLOROPLAST PEP HCO3 -OAA CHLOROPLAST PEP ATP + Pi AMP + PPi
MESOPHYLL CELL BUNDLE SHEATH CELL
RuBP PPdK Rubisco B NAD-ME Pi Malate Aspartate Aspartate Alanine NADH Pyruvate Pyruvate Pyruvate Alanine (NH2) MDH NAD-ME MITOCHONDRION AspAT AlaAT (NH2) AspAT AlaAT PEPC CA
Calvin Cycle CO2 Pyruvate CHLOROPLAST PEP HCO3 -OAA CHLOROPLAST PEP ATP + Pi AMP + PPi
MESOPHYLL CELL BUNDLE SHEATH CELL
RuBP PPdK Rubisco C PEPCK Pi Aspartate Pyruvate Alanine MITOCHONDRION (NH2) AspAT AlaAT PEPC CA OAA Malate NADP+ NADPH MDH Aspartate
OAA PEPCK PEP
ADP ATP CO2 NAD+ NADH Pyruvate Malate NAD-ME H2O O2 ATP ADP Alanine Pyruvate AlaAT (NH2) AspAT CO2 Electron transport Calvin Cycle CO2 Pyruvate CHLOROPLAST PEP HCO3 -OAA CHLOROPLAST PEP ATP + Pi AMP + PPi
MESOPHYLL CELL BUNDLE SHEATH CELL
RuBP PPdK Rubisco C PEPCK Pi Aspartate Pyruvate Alanine MITOCHONDRION (NH2) AspAT AlaAT PEPC CA OAA Malate NADP+ NADPH MDH Aspartate
OAA PEPCK PEP
ADP ATP CO2 NAD+ NADH Pyruvate Malate NAD-ME H2O O2 ATP ADP Alanine Pyruvate AlaAT (NH2) AspAT CO2 Electron transport
Figure I.2. Generalised scheme for the CO2-concentrating mechanisms of the three biochemical subtypes
of C4 photosynthetic pathway (adapted from Kanai & Edwards 1999). In all subtypes, primary CO2
fixation by PEPC in the cytosol of the M cells results in the formation of C4 acids that are transported to
the BS and decarboxylated increasing the CO2 concentration in these cells, where Rubisco and the C3
cycle is located. The C4 cycle is complete once PEP has been regenerated and becomes available for
carboxylation in the mesophyll. In the NADP-ME subtype (A, previous page), malate is the C4 acid
transported to the BS and decarboxylation occurs in the chloroplast of these cells; in the NAD-ME subtype (B, previous page), aspartate is the C4 acid transported to the BS where it is converted into malate
that is decarboxylated in the mitochondria; in the PEPCK subtype (C, this page), aspartate is the main C4
acid transported to the BS where it is converted into oxaloacetate before decarboxylation in the cytosol but, concomitantly, malate is transported from the M chloroplasts to the BS mitochondria where its decarboxylation by NAD-ME provides the energy required for PEPCK (involving use of reducing power for the formation of ATP in the respiratory electron transport system) and contributes to enhance the CO2
concentration available for assimilation through the C3 cycle. Grey-dashed arrows indicate metabolite
transport. Abbreviations used: AlaAT, alanine aminotransferase; AspAT, aspartate aminotransferase; CA, carbonic anhydrase; MDH, malate dehydrogenase; NAD-ME, NAD-malic enzyme; ME, NADP-malic enzyme; PEP, phosphoenolpyruvate; PEPC, PEP carboxylase; PEPCK, PEP carboxykinase; Pi, orthophosphate; PPdK, pyruvate,orthophosphate dikinase; PPi, pyrophosphate; RuBP, ribulose-1,5-bisphosphate.
enzymes involved in the carboxylating and decarboxylating reactions are given in Chapter IV (PEPC and decarboxylating enzymes) and Chapter V (Rubisco).
The coordinate function between the M and BS cells (and thus the C3 and C4 cycles) is
crucial for C4 photosynthesis efficiency. Photosynthesis at high irradiance is limited by the
activities of PEPC and Rubisco (von Caemmerer & Furbank 1999) and a ratio of two between PEPC and Rubisco carboxylation activities is enough to saturate CO2 assimilation. Reduction of
PEPC activity results in low assimilation rates and increased photorespiration rates at high light (Maroco et al. 1998; Bailey et al. 2000), due to the decreased production of C4 acids and,
consequently, of CO2 released in the BS cells. Reduction of Rubisco activity leads to reduced
assimilation rates, increased CO2 concentration in BS cells and increased leakage from the BS
(Furbank et al. 1996; Siebke et al. 1997; von Caemmerer et al. 1997). Because the C4 pathway
has energy costs, associated with the regeneration of PEP (Hatch 1987; Kanai & Edwards 1999), the leakage of CO2 back to the M cells increases the ATP used per mole of CO2 fixed and hence
decreases photosynthetic efficiency. At low irradiance, C4 photosynthesis is mostly limited by
the regeneration of RuBP and PEP, dependent on ATP and NADPH availability (von Caemmerer & Furbank 1999).
The higher water use efficiency (WUE) of C4 plants relative to C3 results from the
combination of an efficient assimilation of CO2 through the C4 pathway, namely because the
CO2-concentrating mechanism limits photorespiration, with lower transpiration rates (Edwards et al. 1985). As reported by Hatch (1987), the WUE in C4 plants corresponds to the double of that
observed in C3 plants and the disparity is likely to increase with temperature, essentially as result
of increased photorespiration in C3 plants.
Photorespiration in C4 plants
Rubisco properties limit the photosynthetic efficiency in C3 plants (Parry et al. 2007), essentially
because the enzyme acts both as a carboxylase and an oxygenase. When CO2 is used as a
substrate for the reaction with RuBP, the product 3-phosphoglycerate (PGA) is metabolised through the Calvin cycle and carbon is assimilated into useful products. Conversely, reaction of RuBP with O2 produces 2-phosphoglycolate (PG) and initiates the process of photorespiration,
which results in the loss of fixed carbon and consumption of energy (Kumarasinghe et al. 1977), thereby decreasing the efficiency of CO2 assimilation. The photorespiratory carbon cycle (Figure
I.3) occurs in three subcellular compartments, the chloroplast, peroxisome and mitochondrion, and results in the release of CO2 and NH3. The remaining carbon is recycled to PGA that
photorespiratory nitrogen cycle (Keys et al. 1978). The involvement of common intermediates in these pathways reflects the great level of integration and interdependence of the photorespiratory carbon oxidation cycle, the photorespiratory nitrogen cycle and the photosynthetic carbon reduction cycle (Keys 1999). The biochemistry of photorespiration and its regulation have been comprehensively reviewed by Leegood et al. (1995).
Glycolate oxidase Amino-transferase Calvin Cycle RuBP PGA Glycolate PG H2O Pi O2 Rubisco P-glycolate oxidase Glycolate H2O2 O2 Glyoxylate Glycine keto acid amino acid Glycine (2) CO2 NH3 Glycine decarboxylase & Serine hydroxymethyl transferase NAD+ NADH Serine Serine Glycerate Glycerate Hydroxypyruvate
CHLOROPLAST PEROXISOME MITOCHONDRION
Glyoxylate Glycine Amino-transferase Reductase NAD+ NADH ADP Glycerate cinase ATP Glycolate oxidase Amino-transferase Calvin Cycle RuBP PGA Glycolate PG H2O Pi O2 Rubisco P-glycolate oxidase Glycolate H2O2 O2 Glyoxylate Glycine keto acid amino acid Glycine (2) CO2 NH3 Glycine decarboxylase & Serine hydroxymethyl transferase NAD+ NADH Serine Serine Glycerate Glycerate Hydroxypyruvate
CHLOROPLAST PEROXISOME MITOCHONDRION
Glyoxylate Glycine Amino-transferase Reductase NAD+ NADH ADP Glycerate cinase ATP
Figure I.3. Simplified scheme of the photorespiratory carbon cycle. The oxygenase reaction of Rubisco
produces PG and PGA. The latter (not shown) is metabolised in the Calvin cycle and PG initiates the photorespiratory cycle. Glutamate and serine are the most common amino group donors for the amination of glyoxylate to glycine in the peroxisome. The subsequent conversion of two glycines into serine in the mitochondrion results in the release of both CO2 and NH3 and the remaining carbon is recycled to PGA
that enters the Calvin cycle in the chloroplast. The regeneration of PGA from glycerate consumes ATP. Grey-dashed arrows indicate metabolite transport. Abbreviations used: PG, 2-phosphoglycolate; PGA, 3-phosphoglycerate; Pi, orthophosphate; RuBP, ribulose-1,5-bisphosphate.
The C4 photosynthetic pathway elevates the CO2 concentration in BS cells and the
resultant high CO2 to O2 ratio offsets the low affinity of Rubisco for CO2 and largely inhibits its
oxygenase activity and, hence, photorespiratory rates in air. Photorespiration rates correspond to about one-quarter of CO2 assimilation rates in C3 species under normal chloroplast conditions at
25ºC (Keys 1986; Sharkey 1988), whilst in C4 plants photorespiration is likely to correspond to
has no effect on PEP carboxylation in both maize and soybean leaf extracts (Bowes & Ogren 1972). Because CO2 and O2 are competing alternative substrates for reaction with RuBP
catalysed by Rubisco, the relative specificity of Rubisco and the concentrations of the gases will determine the flow of carbon between the photorespiratory and the photosynthetic carbon cycles. An increase in photorespiration is therefore expected when the intercellular CO2 decreases, as
may occur under stress conditions promoting stomatal closure.
The CO2-concentrating mechanism has energy costs (Hatch 1987) and the competitive
advantage of C4 plants in relation to C3 depends on the temperature, mostly because higher
temperatures result in increased photorespiration relative to photosynthesis in C3 plants due to
the enhanced ratio of O2 to CO2 in conditions that decrease the solubility of CO2 more than that
of O2 (see Edwards et al. 1985 and references therein).
The estimation of true rates of photosynthesis and photorespiration is difficult because of the complexity of the CO2 and O2 exchanges involved (Sharkey 1988; Keys 1999). The
re-fixation of photorespired CO2 within the leaves (Loreto et al. 1999; Kiirats et al. 2002) further
compromises the estimation of photorespiration through CO2 exchange. Measurement of
photorespiration in C4 plants is especially complex given the level of biochemical and structural
specialization. The mathematical modelling of C4 photosynthesis by von Caemmerer and
Furbank (1999) uses basic equations to represent carbon fluxes in C4 plants. The model assumes
a steady-state balance between the release of CO2 in the BS through the C4 pathway, the
assimilation of CO2 by the C3 cycle and the leakage of CO2 from BS cells. At high irradiance,
CO2 fixation will be limited by PEPC and Rubisco activities and, the CO2 concentration in the
BS cells and, ultimately, the photorespiration rate can be estimated from the analysis of net photosynthesis response to intercellular CO2. Additionally, amino acids can be used as
metabolite markers for the photorespiratory pathway (Foyer et al. 2003) and glycine and serine contents were found to be well correlated with photorespiration (Novitskaya et al. 2002). Further considerations on photorespiration rates in C4 plants are detailed in Chapter III.
DROUGHT STRESS
Drought and water deficit
Stress can be defined as “a deviation from the optimal condition of life” (Larcher 2003) and elicits responses that may be adaptive or cause damage. Water stress occurs when water availability is scarce (water deficit) or excessive (water logging) and becomes likely to affect plant performance. Water deficit is often referred to as drought stress. Drought, in nature, is an event of decreased water availability, resulting from low rainfalls, and is usually associated with high temperatures and irradiance. In climates characterized by hot dry summers, like the Mediterranean, plants are subjected to high temperatures and irradiance and may or not have enough water, but are generally dependent on irrigation. An efficient management of water resources is imperative in order to optimise the use of water in these regions.
Studies in the greenhouse generally attempt to promote conditions as close as possible to those that plants experience in the field, controlling the imposition of stress conditions in order to understand plant responses to the different variables that may affect their growth and productivity. Drought stress, here used as a synonym to water deficit, is one of the major factors limiting plant photosynthetic performance.
Plants may adopt a number of strategies to resist drought stress, including the development of mechanisms of drought avoidance or drought tolerance. In the first case, the hydration of the tissues is maintained through processes that minimize water loss or increase water uptake from the soil, whilst in the latter one the physiological and biochemical parameters are maintained when tissue dehydration occurs (Chaves 1991; Bray et al. 2000; Larcher 2003; Blum 2005). Drought avoidance strategies are much dependent on the early signals triggered at the root level in response to the drying soil (Chaves & Oliveira 2004). These signals warn the plant to the decreased water status in the soil and allow the development of a stress response in order to better cope with the unfavourable environment. The regulation of plant growth and development in response to drying soil often involves chemical signalling that may concomitantly result in stomatal closure to avoid excessive water loss from the tissues (Davies et al. 2005). In fact, both stomatal closure (Schulze 1986) and shoot growth inhibition (Kramer 1983) occur at the earliest stages of water deficit and are likely to be induced by stress sensors or through signal transduction pathways triggered in response to stress (Bartels & Sunkar 2005). The long-distance signalling, involving the transit of molecules (like the hormone ABA and other chemical regulators) from the root to the shoot and vice-versa, provide the plant with a
