*3.2.2.2. Plant architecture*

An obvious target for increasing the source strength is the production of photoassimilates during photosynthesis. In this respect, research has been undertaken to introduce a more efficient, C4-like photosynthesis in C3 plants [58]. These last workers indicated that the introduction of single C4 enzymes (i.e., phosphoenol pyruvate carboxylase, PEPC, and pyruvate orthophosphate dikinase, PPDK) in C3 plants has until now not generated improve‐ ment in photoassimilate accumulation. This is probably attributable to disturbances in the fluxes of C4 intermediates for metabolic pathways excluding the C4 cycle. Notably in rice, the joined expression of two C4 cycle enzymes was found to raise photosynthetic ability up to 35% and grain yield up to 22%. In this research, the maize genes were transferred to the rice genome together with their corresponding promoters, which might have turned out in a superior spatial and temporal expression of the C4 cycle enzymes than the more expected expression of transgenes by using constitutive promoters. Additionally, work in transgenic tobacco has also showed that increased levels of fructose-1, 6-bisphosphatase (FBPase) and sedoheptu‐ lose-1,7-bisphosphatase (SBPase), two Calvin cycle enzymes, significantly increased dry

Besides the above strategies based on C4 photosynthesis, other approaches have been taken to improve the efficiency of photosynthetic C assimilation. One of these strategies is focused on the enzyme RUBISCO ACTIVASE, a key regulator of RUBISCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase essential component of the photosynthetic process of fixing CO2 into organic C) activity [60]. Transgenic Arabidopsis plants expressing a heat-tolerant version of RUBISCO ACTIVASE showed a significant improvement in photosynthesis and leaf growth when exposed to heat. Other efforts to improve the photosynthetic efficiency of plants have been focused on: i) increasing electron flow by overexpressed CYTOCHROME C6 (CYTC6), a protein involved in the photosynthetic electron transport chain [61]; ii) engineering new pathways into the chloroplast that bypass photorespiration and release CO2 directly into the chloroplast stroma [62] ; and iii) using various 'add-ons' or CO2-concentrating mechanisms (CCMs) to elevate CO2 levels in the vicinity of RUBISCO. These CCMs were pursued by improving mesophyll CO2 conductance via overexpressing aquaporin [63] or stomatal CO2 conductance via manipulating stomatal characteristics [64]. In this respect, more recently Lin and co-workers [65] have successfully engineered tobacco plants containing a functioning RUBISCO from a cyanobacterium. The cyanobacterial enzyme has a greater catalytic rate than any 'C3' plant. The lines generated in this research open the way for future addition of the remaining components of the cyanobacterial CCM, an important step towards enhancing photosynthetic efficiency and improving crop yields. Furthermore, it has been shown that the triose-phosphate/phosphate translocator (TPT) strongly limits photosynthesis under high CO2 conditions [65]. The TPT provides a regulatory link between CO2 assimilation and cytosolic C metabolism. In this context an approach for increasing plant yield was performed by overexpressing sucrose transporters in sink cells, thereby enhancing sink demand and inducing an increase in photosynthesis and assimilates export. When overexpresssing a potato sucrose symporter (*StSUT1*) in storage parenchyma cells of pea seeds, there was enhanced sucrose influx into cotyledons and greater cotyledon growth rates [67]. In addition, it has been shown that enhancement of sucrose synthase activity represents a useful strategy for increas‐ ing starch accumulation and yield in potato tubers. In a future world of higher CO2 concen‐

weight [59].

116 Plants for the Future

Considerable progress has been realized over the past decade in revealing the molecular mechanisms that underlie the formation of plant architecture. Dissection of plant architecture with plant morphological mutants enabled plant scientists to gain insides into various aspects affecting plant architecture as exemplified by the shot apical meristem (SAM) maintenance and differentiation, the initiation of axillary meristems (AMs), the formation and outgrowth of axillary buds, the elongation of stems, and the number or angle of branches (tillers) [68].

Studies carried out in model plants (i.e., Arabidopsis and petunia) and crop plants (e.g., tomato, maize, and rice) served as platforms for understanding the molecular basis of plant architec‐ ture. In this respect, RNA interference (RNAi) technology was used to improve crop produc‐ tivity by modifying plant architecture. For example, in rice over-expression or suppression of the *OsPIN1* (a member of the gene family of auxin transporters in plants) expression through a transgenic approach resulted in changes of tiller numbers and shoot/root ratio, suggesting that *OsPIN1* played a role in auxin-dependent adventitious root emergence and tillering [69]. In general, these studies have provided insights on the molecular mechanisms affecting each specific aspect that collectively specializes a particular type of plant architecture, among which the signaling pathways that regulate plant height and control the maintenance and differen‐ tiation of the SAM have been particularly well analyzed. Although our current understanding of how a particular plant specializes its architecture is still poor, the findings obtained so far in studying plant architecture have already allowed us to open a way for optimizing the plant architecture of crops by molecular design and improving biomass productivity.

#### *3.2.2.3. Regulation of plant biomass production*

In plants, the potential to improve biomass production has not yet been largely investigated. This is because the conventional breeding of crop plants (e.g., maize, rice, wheat, and soybean) has been centered on the selection of the superior grain-yield traits. However, current molec‐ ular and genetic research has singled out a number of regulators affecting plant biomass production. A diagram depicting various factors affecting plant biomass production reported by Demura and Ye [70] is given in Figure 7. It is evident from this graphical representation that an increase in biomass yield could be achieved by an enhancement of the vegetative apical meristem activity by KNOX proteins (i.e., KNOX are homeodomain TFs) and of the vascular cambium activity by KNOX and cytokinin, an inhibition of the transition into reproductive growth by activating FLC (protein encoded by the *FLOWERING LOCUS C,FLC,* affecting flowering time) and by repressing SOC1/FUL/FT (encoded respectively by *FLOWERING LOCUS T, FT*, and *SUPPRESSOR OF OVEREXPRESSION OF CONSTAxlink (SOC1*), both involved in regulation of the indeterminacy of meristems and the longevity of plants), an increased cell elongation by gibberellin, an elevated photosynthetic efficiency, and an increase in secondary cell wall biosynthesis. An improved cellulose digestibility by modifying cell wall properties could contribute to increased biofuel production. Therefore, flowering-time genes could potentially be manipulated for generation of grass crops with increased biomass yield. Overexpression of some members of NAC family (a plant-specific family of TFs) also induces increased biomass production. Arabidopsis transgenic lines overexpressing *NAC1* are bigger, with larger leaves, thicker stems, and more abundant roots compared with control plants [71]. Similarly, overexpression in Arabidopsis of another NAC-domain transcription factor, ATAF2, leads to increased biomass, mainly by production of bigger leaves with larger cells [72].

Additionally it is obvious that genes regulating cell number and organ size in plants could potentially contribute to yield increases. For example in maize, Guo et al. [73] have isolated and described a family of genes termed *Cell Number Regulators (CNRs)*. Insight into their function was achieved by ectopically expressing one of this members, namely *CNR1.* It was shown that *CNR1* reduced overall plant size when ectopically overexpressed, while plant and organ size increased when its expression was co-suppressed or silenced. Leaf epidermal cell counts showed that the increased or decreased transgenic plant and organ size was due to changes in cell number, not cell size supporting the idea that *CNR*s function as cell number regulators in maize. This suggests the potential for application to crop improvement via generation of more vigorous and productive plants.

#### *3.2.2.4. Phytohormones*

Phytohormones are known to play important roles in plant growth and development, including the regulation of meristematic activities and cell elongation, both of which are crucial for biomass yield. For example, the plant hormones auxin and brassinosteroids (BRs) are important regulators of plant growth, stimulating both cell division and cell elongation. Arabidopsis plants that are unable to synthesize or perceive BRs are dwarfs with rounded leaves and reduced pollen fertility that show sizeable delayed flowering time and leaf senescence [74]. STEROID 22a HYDROXYLASE, encoded by the *DWF4* gene, is a key enzyme for BR biosynthesis. Overexpression of *DWF4* in *A. thaliana* produced plants that grew 35–47% taller and produced 33% more seed. The rice mutant *dwf4-1* had depressed levels of BRs and an ideotype characterized by slight dwarfism and erect leaves. Although individual *dwf4-1* plants had reduced biomass yield, their ideotype allowed high-density planting that led to increased grain yield per unit area [75].

A way by which auxin controls final plant organ size is through the transcription factor *ARGOS (AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE*) which acts upstream of *AINTE‐* Genetic Strategies to Enhance Plant Biomass Yield and Quality-Related Traits for Bio-Renewable Fuel and... http://dx.doi.org/10.5772/61005 119

that an increase in biomass yield could be achieved by an enhancement of the vegetative apical meristem activity by KNOX proteins (i.e., KNOX are homeodomain TFs) and of the vascular cambium activity by KNOX and cytokinin, an inhibition of the transition into reproductive growth by activating FLC (protein encoded by the *FLOWERING LOCUS C,FLC,* affecting flowering time) and by repressing SOC1/FUL/FT (encoded respectively by *FLOWERING LOCUS T, FT*, and *SUPPRESSOR OF OVEREXPRESSION OF CONSTAxlink (SOC1*), both involved in regulation of the indeterminacy of meristems and the longevity of plants), an increased cell elongation by gibberellin, an elevated photosynthetic efficiency, and an increase in secondary cell wall biosynthesis. An improved cellulose digestibility by modifying cell wall properties could contribute to increased biofuel production. Therefore, flowering-time genes could potentially be manipulated for generation of grass crops with increased biomass yield. Overexpression of some members of NAC family (a plant-specific family of TFs) also induces increased biomass production. Arabidopsis transgenic lines overexpressing *NAC1* are bigger, with larger leaves, thicker stems, and more abundant roots compared with control plants [71]. Similarly, overexpression in Arabidopsis of another NAC-domain transcription factor, ATAF2, leads to increased biomass, mainly by production of bigger leaves with larger cells [72].

Additionally it is obvious that genes regulating cell number and organ size in plants could potentially contribute to yield increases. For example in maize, Guo et al. [73] have isolated and described a family of genes termed *Cell Number Regulators (CNRs)*. Insight into their function was achieved by ectopically expressing one of this members, namely *CNR1.* It was shown that *CNR1* reduced overall plant size when ectopically overexpressed, while plant and organ size increased when its expression was co-suppressed or silenced. Leaf epidermal cell counts showed that the increased or decreased transgenic plant and organ size was due to changes in cell number, not cell size supporting the idea that *CNR*s function as cell number regulators in maize. This suggests the potential for application to crop improvement via

Phytohormones are known to play important roles in plant growth and development, including the regulation of meristematic activities and cell elongation, both of which are crucial for biomass yield. For example, the plant hormones auxin and brassinosteroids (BRs) are important regulators of plant growth, stimulating both cell division and cell elongation. Arabidopsis plants that are unable to synthesize or perceive BRs are dwarfs with rounded leaves and reduced pollen fertility that show sizeable delayed flowering time and leaf senescence [74]. STEROID 22a HYDROXYLASE, encoded by the *DWF4* gene, is a key enzyme for BR biosynthesis. Overexpression of *DWF4* in *A. thaliana* produced plants that grew 35–47% taller and produced 33% more seed. The rice mutant *dwf4-1* had depressed levels of BRs and an ideotype characterized by slight dwarfism and erect leaves. Although individual *dwf4-1* plants had reduced biomass yield, their ideotype allowed high-density planting that led to

A way by which auxin controls final plant organ size is through the transcription factor *ARGOS (AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE*) which acts upstream of *AINTE‐*

generation of more vigorous and productive plants.

increased grain yield per unit area [75].

*3.2.2.4. Phytohormones*

118 Plants for the Future

**Figure 7.** Schematic representation indicating several factors influencing plant biomass yield (Redrawn from Demura and Ye, 2010, [70]). **Figure 7.** Schematic representation indicating several factors influencing plant biomass yield (Redrawn from Demura and Ye, 2010, [70]).

*GUMENTA (ANT*), a member of the AP2/ERF family of TFs [76]. For both *ANT* and *ARGOS*, transgenic plants overexpression resulted in an increased plant and organ size. These last genes promote growth through prolonging the cell proliferation period (but not the rate), and the final organ size change is mainly due to an increase in cell number, not cell size. Notably, Arabidopsis *ARGOS-LIKE*, another growth promoting factor, enlarges organ size by enlarging cell size not cell number. *EBP1,* a putative Arabidopsis ortholog of human *ErbB-3* epidermal growth factor receptor binding protein, is another key regulator promoting plant and organ growth. In contrast with ARGOS and ANT, transgenically overexpressing *EBP1* accelerates plant growth and development by simultaneously stimulating cell growth, proliferation, and development, resulting in both increased cell number and cell size [77]. For all three of these growth-promoting genes, not only does transgenically increasing expression promote plant and organ growth, but also down-regulation of gene expression or loss-of-function via mutation reduces the plant and organ growth. Among these key players are growth repressors, an example is Arabidopsis *AUXIN RESPONSE FACTOR2* (*ARF2*). Loss of *ARF2* function causes enhanced organ size, such as thicker stems and larger seeds, floral organs, and leaves [78]. Physiological studies have suggested synergistic roles for auxin and BRs in cell elongation and genome-wide microarray expression studies identified a large number of genes that respond to both auxin and BRs [79]. Integration of auxin and BR signaling has been suggested to occur at the level of ARFs because the auxin-response element (TGTCTC) is enriched in the promoters of BR-responsive genes [80].

There is information indicating that cytokinins also promote organ growth by stimulating cell proliferation with cytokinin depletion or overproduction resulting in smaller or larger leaves and flowers, respectively [76]. Likewise, gibberellins promote growth through expansion and/ or proliferation, acting to repress the activity of the growth-restraining DELLA proteins; DELLA factors may be important in adjusting growth in response to environmental influences. In addition to these classical phytohormones, a novel mobile signal whose synthesis depends on the related cytochrome P450 enzymes KLUH/CYP78A5 and CYP78A7 promotes leaf and floral-organ growth. Investigations on chimaeric plants indicate that the presumed signal is integrated across flowers, suggesting that it may be used to coordinate growth within and between individual organs [76].

In conclusion, it is worth noting that a better understanding of genes affecting hormonal metabolism and signaling should help to design strategies to increase plant growth and organ size and, ultimately, crop yield.

#### *3.2.2.5. Enhancing sink strength*

To increase sink strength, credible biochemical targets are represented by enzymes directly or indirectly involved in the conversion of sucrose into starch. One approach to increase sink strength has been devoted to modify the adenylate pools in potatoes; adenylate levels was found to be relevant for starch content in their tubers [81]. In this respect, it was shown that down-regulation of the plastidial isoform of adenylate kinase induced a 60% upgrade of starch in potatoes and, interestingly, a 39% gain in tuber production [82].

Other approaches for yield improvement are more specifically directed towards starch synthesis. Many of these attempts showed only limited success [83]. On the contrary, positive results were obtained in different plant species by acting on AGPase that catalyses a ratelimiting step in the starch biosynthetic pathway. As mentioned above, these enzymes, subject to allosteric control by orthophosphate (Pi ) and 3-phosphoglycerate (PG) effectors, have received the most attention [84]. For example, in maize overexpression of wild-type *Sh2* (that encodes the endosperm large subunit of AGPase suscettible to Pi inhibition) and *Bt-2* (*Bt2*, which encodes the endosperm small subunit of AGPase stimulated by PG) increased individ‐ ual seed weight to 15% by increasing starch content [85]. More recently, Hannah and coworkers [86], by studying the expression of a transgenic form (*HS33/Rev6 Sh*2) of AGPase with enhanced heat stability and reduced Pi inhibition, reported an increased maize yield up to 64%. Interestingly, as previously found in wheat and rice, this transgene increases maize yield by increasing seed number. Moreover. genetic, physiological, and molecular data provided by Hannah et al. [86] point to *HS33/Rev6 Sh2* expression in maternal tissue, rather than in the seed, as the cause of enhanced seed number. Therefore, these workers concluded that the transgene does not increase ovary number; rather, it increases the probability that a seed will develop. Furthermore, the addition of allosterically altered AGPases and increased Arabidopsis leaf transitory starch turnover improved its growth characteristics [87] and increased fresh weights of aerial parts of lettuce plants ([88].

## *3.2.2.6. Other strategies*

and organ growth, but also down-regulation of gene expression or loss-of-function via mutation reduces the plant and organ growth. Among these key players are growth repressors, an example is Arabidopsis *AUXIN RESPONSE FACTOR2* (*ARF2*). Loss of *ARF2* function causes enhanced organ size, such as thicker stems and larger seeds, floral organs, and leaves [78]. Physiological studies have suggested synergistic roles for auxin and BRs in cell elongation and genome-wide microarray expression studies identified a large number of genes that respond to both auxin and BRs [79]. Integration of auxin and BR signaling has been suggested to occur at the level of ARFs because the auxin-response element (TGTCTC) is enriched in the

There is information indicating that cytokinins also promote organ growth by stimulating cell proliferation with cytokinin depletion or overproduction resulting in smaller or larger leaves and flowers, respectively [76]. Likewise, gibberellins promote growth through expansion and/ or proliferation, acting to repress the activity of the growth-restraining DELLA proteins; DELLA factors may be important in adjusting growth in response to environmental influences. In addition to these classical phytohormones, a novel mobile signal whose synthesis depends on the related cytochrome P450 enzymes KLUH/CYP78A5 and CYP78A7 promotes leaf and floral-organ growth. Investigations on chimaeric plants indicate that the presumed signal is integrated across flowers, suggesting that it may be used to coordinate growth within and

In conclusion, it is worth noting that a better understanding of genes affecting hormonal metabolism and signaling should help to design strategies to increase plant growth and organ

To increase sink strength, credible biochemical targets are represented by enzymes directly or indirectly involved in the conversion of sucrose into starch. One approach to increase sink strength has been devoted to modify the adenylate pools in potatoes; adenylate levels was found to be relevant for starch content in their tubers [81]. In this respect, it was shown that down-regulation of the plastidial isoform of adenylate kinase induced a 60% upgrade of starch

Other approaches for yield improvement are more specifically directed towards starch synthesis. Many of these attempts showed only limited success [83]. On the contrary, positive results were obtained in different plant species by acting on AGPase that catalyses a ratelimiting step in the starch biosynthetic pathway. As mentioned above, these enzymes, subject

received the most attention [84]. For example, in maize overexpression of wild-type *Sh2* (that

which encodes the endosperm small subunit of AGPase stimulated by PG) increased individ‐ ual seed weight to 15% by increasing starch content [85]. More recently, Hannah and coworkers [86], by studying the expression of a transgenic form (*HS33/Rev6 Sh*2) of AGPase with

Interestingly, as previously found in wheat and rice, this transgene increases maize yield by

) and 3-phosphoglycerate (PG) effectors, have

inhibition, reported an increased maize yield up to 64%.

inhibition) and *Bt-2* (*Bt2*,

in potatoes and, interestingly, a 39% gain in tuber production [82].

encodes the endosperm large subunit of AGPase suscettible to Pi

to allosteric control by orthophosphate (Pi

enhanced heat stability and reduced Pi

promoters of BR-responsive genes [80].

120 Plants for the Future

between individual organs [76].

size and, ultimately, crop yield.

*3.2.2.5. Enhancing sink strength*

An approach to remodel plant growth is to vary the expression of cell cycle-related genes. Cockcroft et al. [89], by overexpressing the Arabidopsis *CYCD2* gene in tobacco, obtained plants that were 35% higher in comparison to controls. These transgenic plants also manifested normal cell and meristem size associated with an exalted overall growth rates, an enhanced rate of leaf initiation, and a faster rate of growth at all stages of plant development considered in the experiment. Similarly, the overexpression of another Arabidopsis D-type cyclin, *CYCD3;1*, a rate-limiting gene affecting the G1/S transition phase, was found to promote ectopic cell divisions, generating leaves with more but smaller cells [90]). On the contrary, the ablation of *ABAP1*, an Arabidopsis gene that controls the cell proliferation rate by limiting mitotic DNA replication, resulted in larger leaves containing more cells [91]. Further studies on developing leaves revealed that during early leaf development, cell division rates were smaller in *ABAP1*-overexpressing plants in comparison with controls. On the other hand, cell division was faster in plants possessing a defective copy of *ABAP1* gene. Another gene whose variation increases biomass is *CDC27a* [92]. This gene encodes a protein that is a component of the ligase Anaphase-promoting Complex (APC). Overexpression of Arabidopsis *CDC27a* in tobacco produced plants that were up to 30% higher at flowering time, with slight alterations of apical meristems [92]. These last authors concluded that it is likely that the growth promo‐ tion mechanism observed in *CDC27a* overexpressing plants is due to the APC, rather than CDC27a protein itself, because the overexpression of at least two other subunits of the APC displays similar enhanced growth.

Another mechanism promoting cell proliferation and ultimately organ growth involves TFs of the *TCP* (*TEOSINTE BRANCHED1, CYCLOIDEA, PCFs) and GROWTH-REGULATING FACTOR (GRF*) classes, two redundant multi-gene families in Arabidopsis. The importance of the TCP family in growth control became evident from the *cincinnata (cin*) mutant in snapdra‐ gon (Antirrhinum) and the jaw-D activation-tagged mutant of Arabidopsis [93,94]. In these mutants, leaves overgrow to a highly-wrinkled shape because of excess cell proliferation particularly at the leaf margins. It was found that *CIN* encodes a member of the TCP family and in *cin* mutants, the size of the proliferative region at the leaf base appears enlarged and its distal boundary is concave. In this respect cells at the leaf margin still proliferate at positions where cells in the center have already arrested proliferation. In the *jaw-D* mutant, overexpres‐ sion of the microRNA miR319a down-regulates five genes of the TCP family. Down-regulation of another three gene family members causes even more severe phenotypes of overprolifera‐ tion in leaves, while miR319-resistant versions of TCPs and loss-of-function mutations in miR319a reduce organ size and cause premature cell differentiation [93, 94]. In fact, promoting cell differentiation has been proposed as the primary function of TCPs, rather than directly arresting proliferation [95].

A novel gene regulation mechanism was recently discovered in metazoans, the RNA silencing or RNAi, or microRNA (miRNAs) [96]. Rodriguez et al. [97] found that overexpression of miR396 in Arabidopsis had a negative impact on cell proliferation in developing leaves through the repression of GRF activity and a decrease in the expression of cell cycle genes. Accordingly, disruption of the recognition of *GRF2* by miR396 resulted in larger leaves with more cells than control plants. Similarly in Arabidopsis, it was reported that over-expression of miR156 promotes a vegetative-phase transition delay and an enhancement in leaf initiation rate [98], while over-expression of miR172 induces adult leaf traits and flowering [99]. The regulation of both miRNAs is closely connected as their expression is influenced by age, temperature, and light acting in a contrasting way [100]. At a molecular level, miR156 acts through the down regulation of genes coding for the *SQUAMOSA PROMOTER BINDING-LIKE (SPL*) and the *AP2-like transcription factor*, respectively, are subjected to feedback regula‐ tion by their targets [101].

Recently, miRNAs have also emerged as key regulators of phytohormone response pathways *in planta* by affecting their metabolism, distribution, and perception [100,102] have demon‐ strated that gibberellic acid (GA) promotes flowering in Arabidopsis through a miR156 dependent pathway, indicating a connection between miRNA and phytohormone signaling pathways in the control of shoot development. In general, it appears evident from this data that miRNAs as growth regulators are interesting targets for gene manipulation for improving biomass yield.
